Gene disruptions, compositions and methods relating thereto

ABSTRACT

The present invention relates to transgenic animals, as well as compositions and methods relating to the characterization of gene function. Specifically, the present invention provides transgenic mice comprising disruptions in PRO226, PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196, PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161 genes. Such in vivo studies and characterizations may provide valuable identification and discovery of therapeutics and/or treatments useful in the prevention, amelioration or correction of diseases or dysfunctions associated with gene disruptions such as neurological disorders; cardiovascular, endothelial or angiogenic disorders; eye abnormalities; immunological disorders; oncological disorders; bone metabolic abnormalities or disorders; lipid metabolic disorders; or developmental abnormalities.

RELATED APPLICATIONS

This application is a US national stage continuation applicationclaiming priority under 35 USC §371 of international applicationPCT/US2006/027777, filed Jul. 18, 2006, which claims priority under 35USC §119 to US Provisional Application 60/708,312 filed Aug. 15, 2005.

FIELD OF THE INVENTION

The present invention relates to compositions, including transgenic andknockout animals and methods of using such compositions for thediagnosis and treatment of diseases or disorders.

BACKGROUND OF THE INVENTION

Extracellular proteins play important roles in, among other things, theformation, differentiation and maintenance of multicellular organisms.The fate of many individual cells, e.g., proliferation, migration,differentiation, or interaction with other cells, is typically governedby information received from other cells and/or the immediateenvironment. This information is often transmitted by secretedpolypeptides (for instance, mitogenic factors, survival factors,cytotoxic factors, differentiation factors, neuropeptides, and hormones)which are, in turn, received and interpreted by diverse cell receptorsor membrane-bound proteins. These secreted polypeptides or signalingmolecules normally pass through the cellular secretory pathway to reachtheir site of action in the extracellular environment.

Secreted proteins have various industrial applications, including aspharmaceuticals, diagnostics, biosensors and bioreactors. Most proteindrugs available at present, such as thrombolytic agents, interferons,interleukins, erythropoietins, colony stimulating factors, and variousother cytokines, are secretory proteins. Their receptors, which aremembrane proteins, also have potential as therapeutic or diagnosticagents. Efforts are being undertaken by both industry and academia toidentify new, native secreted proteins. Many efforts are focused on thescreening of mammalian recombinant DNA libraries to identify the codingsequences for novel secreted proteins. Examples of screening methods andtechniques are described in the literature [see, for example, Klein etal., Proc. Natl. Acad. Sci. 93:7108-7113 (1996); U.S. Pat. No.5,536,637)].

Membrane-bound proteins and receptors can play important roles in, amongother things, the formation, differentiation and maintenance ofmulticellular organisms. The fate of many individual cells, e.g.,proliferation, migration, differentiation, or interaction with othercells, is typically governed by information received from other cellsand/or the immediate environment. This information is often transmittedby secreted polypeptides (for instance, mitogenic factors, survivalfactors, cytotoxic factors, differentiation factors, neuropeptides, andhormones) which are, in turn, received and interpreted by diverse cellreceptors or membrane-bound proteins. Such membrane-bound proteins andcell receptors include, but are not limited to, cytokine receptors,receptor kinases, receptor phosphatases, receptors involved in cell-cellinteractions, and cellular adhesion molecules like selectins andintegrins. For instance, transduction of signals that regulate cellgrowth and differentiation is regulated in part by phosphorylation ofvarious cellular proteins. Protein tyrosine kinases, enzymes thatcatalyze that process, can also act as growth factor receptors. Examplesinclude fibroblast growth factor receptor and nerve growth factorreceptor.

Membrane-bound proteins and receptor molecules have various industrialapplications, including as pharmaceutical and diagnostic agents.Receptor immuno-adhesions, for instance, can be employed as therapeuticagents to block receptor-ligand interactions. The membrane-boundproteins can also be employed for screening of potential peptide orsmall molecule inhibitors of the relevant receptor/ligand interaction.

Efforts are being undertaken by both industry and academia to identifynew, native receptor or membrane-bound proteins. Many efforts arefocused on the screening of mammalian recombinant DNA libraries toidentify the coding sequences for novel receptor or membrane-boundproteins.

Given the importance of secreted and membrane-bound proteins inbiological and disease processes, in vivo studies and characterizationsmay provide valuable identification and discovery of therapeutics and/ortreatments useful in the prevention, amelioration or correction ofdiseases or dysfunctions. In this regard, genetically engineered micehave proven to be invaluable tools for the functional dissection ofbiological processes relevant to human disease, including immunology,cancer, neuro-biology, cardiovascular biology, obesity and many others.Gene knockouts can be viewed as modeling the biological mechanism ofdrug action by presaging the activity of highly specific antagonists invivo. Knockout mice have been shown to model drug activity; phenotypesof mice deficient for specific pharmaceutical target proteins canresemble the human clinical phenotype caused by the correspondingantagonist drug. Gene knockouts enable the discovery of the mechanism ofaction of the target, the predominant physiological role of the target,and mechanism-based side-effects that might result from inhibition ofthe target in mammals. Examples of this type include mice deficient inthe angiotensin converting enzyme (ACE) [Esther, C. R. et al., Lab.Invest., 74:953-965 (1996)] and cyclooxygenase-1 (COX1) genes[Langenbach, R. et al., Cell, 83:483-492 (1995)]. Conversely, knockingthe gene out in the mouse can have an opposite phenotypic effect to thatobserved in humans after administration of an agonist drug to thecorresponding target. Examples include the erythropoietin knockout [Wu,C. S. et al., Cell, 83:59-67 (1996)], in which a consequence of themutation is deficient red blood cell production, and the GABA(A)-R-β3knockout [DeLorey, T. M., J. Neurosci., 18:8505-8514 (1998)], in whichthe mutant mice show hyperactivity and hyper-responsiveness. Both thesephenotypes are opposite to the effects of erythropoietin andbenzodiazepine administration in humans. A striking example of a targetvalidated using mouse genetics is the ACC2 gene. Although the human ACC2gene had been identified several years ago, interest in ACC2 as a targetfor drug development was stimulated only recently after analysis of ACC2function using a knockout mouse. ACC2 mutant mice eat more than theirwild-type littermates, yet burn more fat and store less fat in theiradipocytes, making this enzyme a probable target for chemical antagonismin the treatment of obesity [Abu-Elheiga, L. et al., Science,291:2613-2616 (2001)].

In the instant application, mutated gene disruptions have resulted inphenotypic observations related to various disease conditions ordysfunctions including: CNS/neurological disturbances or disorders suchas anxiety; eye abnormalities and associated diseases; cardiovascular,endothelial or angiogenic disorders including atherosclerosis; abnormalmetabolic disorders including diabetes and dyslipidemias associated withelevated serum triglycerides and cholesterol levels; immunological andinflammatory disorders; oncological disorders; bone metabolicabnormalities or disorders such as arthritis, osteoporosis andosteopetrosis; or a developmental disease such as embryonic lethality.

SUMMARY OF THE INVENTION A. Embodiments

The invention provides an isolated nucleic acid molecule comprising anucleotide sequence that encodes a PRO226, PRO257, PRO268, PRO290,PRO36006, PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125,PRO1134, PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419,PRO1433, PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193,PRO4341, PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989,PRO5737, PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852,PRO9873, PRO10196, PRO34778, PRO20233, PRO21956, PRO57290, PRO38465,PRO38683 or PRO85161 polypeptide.

In one aspect, the isolated nucleic acid molecule comprises a nucleotidesequence having at least about 80% nucleic acid sequence identity,alternatively at least about 81% nucleic acid sequence identity,alternatively at least about 82% nucleic acid sequence identity,alternatively at least about 83% nucleic acid sequence identity,alternatively at least about 84% nucleic acid sequence identity,alternatively at least about 85% nucleic acid sequence identity,alternatively at least about 86% nucleic acid sequence identity,alternatively at least about 87% nucleic acid sequence identity,alternatively at least about 88% nucleic acid sequence identity,alternatively at least about 89% nucleic acid sequence identity,alternatively at least about 90% nucleic acid sequence identity,alternatively at least about 91% nucleic acid sequence identity,alternatively at least about 92% nucleic acid sequence identity,alternatively at least about 93% nucleic acid sequence identity,alternatively at least about 94% nucleic acid sequence identity,alternatively at least about 95% nucleic acid sequence identity,alternatively at least about 96% nucleic acid sequence identity,alternatively at least about 97% nucleic acid sequence identity,alternatively at least about 98% nucleic acid sequence identity andalternatively at least about 99% nucleic acid sequence identity to (a) aDNA molecule encoding a PRO226, PRO257, PRO268, PRO290, PRO36006,PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125, PRO1134,PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433,PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341,PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737,PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873,PRO10196, PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 orPRO85161 polypeptide having a full-length amino acid sequence asdisclosed herein, an amino acid sequence lacking the signal peptide asdisclosed herein, an extracellular domain of a transmembrane protein,with or without the signal peptide, as disclosed herein or any otherspecifically defined fragment of the full-length amino acid sequence asdisclosed herein, or (b) the complement of the DNA molecule of (a).

In other aspects, the isolated nucleic acid molecule comprises anucleotide sequence having at least about 80% nucleic acid sequenceidentity, alternatively at least about 81% nucleic acid sequenceidentity, alternatively at least about 82% nucleic acid sequenceidentity, alternatively at least about 83% nucleic acid sequenceidentity, alternatively at least about 84% nucleic acid sequenceidentity, alternatively at least about 85% nucleic acid sequenceidentity, alternatively at least about 86% nucleic acid sequenceidentity, alternatively at least about 87% nucleic acid sequenceidentity, alternatively at least about 88% nucleic acid sequenceidentity, alternatively at least about 89% nucleic acid sequenceidentity, alternatively at least about 90% nucleic acid sequenceidentity, alternatively at least about 91% nucleic acid sequenceidentity, alternatively at least about 92% nucleic acid sequenceidentity, alternatively at least about 93% nucleic acid sequenceidentity, alternatively at least about 94% nucleic acid sequenceidentity, alternatively at least about 95% nucleic acid sequenceidentity, alternatively at least about 96% nucleic acid sequenceidentity, alternatively at least about 97% nucleic acid sequenceidentity, alternatively at least about 98% nucleic acid sequenceidentity and alternatively at least about 99% nucleic acid sequenceidentity to (a) a DNA molecule comprising the coding sequence of afull-length PRO226, PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365,PRO382, PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281,PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550,PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369,PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993,PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196,PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161polypeptide cDNA as disclosed herein, the coding sequence of a PRO226,PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382, PRO444,PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343, PRO1379,PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571, PRO1572,PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381, PRO4407,PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017, PRO7174,PRO9744, PRO9821, PRO9852, PRO9873, PRO10196, PRO34778, PRO20233,PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161 polypeptide lackingthe signal peptide as disclosed herein, the coding sequence of anextracellular domain of a transmembrane PRO226, PRO257, PRO268, PRO290,PRO36006, PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125,PRO1134, PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419,PRO1433, PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193,PRO4341, PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989,PRO5737, PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852,PRO9873, PRO10196, PRO34778, PRO20233, PRO21956, PRO57290, PRO38465,PRO38683 or PRO85161 polypeptide, with or without the signal peptide, asdisclosed herein or the coding sequence of any other specificallydefined fragment of the full-length amino acid sequence as disclosedherein, or (b) the complement of the DNA molecule of (a).

In a further aspect, the invention concerns an isolated nucleic acidmolecule comprising a nucleotide sequence having at least about 80%nucleic acid sequence identity, alternatively at least about 81% nucleicacid sequence identity, alternatively at least about 82% nucleic acidsequence identity, alternatively at least about 83% nucleic acidsequence identity, alternatively at least about 84% nucleic acidsequence identity, alternatively at least about 85% nucleic acidsequence identity, alternatively at least about 86% nucleic acidsequence identity, alternatively at least about 87% nucleic acidsequence identity, alternatively at least about 88% nucleic acidsequence identity, alternatively at least about 89% nucleic acidsequence identity, alternatively at least about 90% nucleic acidsequence identity, alternatively at least about 91% nucleic acidsequence identity, alternatively at least about 92% nucleic acidsequence identity, alternatively at least about 93% nucleic acidsequence identity, alternatively at least about 94% nucleic acidsequence identity, alternatively at least about 95% nucleic acidsequence identity, alternatively at least about 96% nucleic acidsequence identity, alternatively at least about 97% nucleic acidsequence identity, alternatively at least about 98% nucleic acidsequence identity and alternatively at least about 99% nucleic acidsequence identity to (a) a DNA molecule that encodes the same maturepolypeptide encoded by any of the human protein cDNAs deposited with theATCC as disclosed herein, or (b) the complement of the DNA molecule of(a).

Another aspect of the invention provides an isolated nucleic acidmolecule comprising a nucleotide sequence encoding a PRO226, PRO257,PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382, PRO444, PRO705,PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343, PRO1379, PRO1380,PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571, PRO1572, PRO1759,PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381, PRO4407, PRO4425,PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017, PRO7174, PRO9744,PRO9821, PRO9852, PRO9873, PRO10196, PRO34778, PRO20233, PRO21956,PRO57290, PRO38465, PRO38683 or PRO85161 polypeptide which is eithertransmembrane domain-deleted or transmembrane domain-inactivated, or iscomplementary to such encoding nucleotide sequence, wherein thetransmembrane domain(s) of such polypeptide are disclosed herein.Therefore, soluble extracellular domains of the herein described PRO226,PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382, PRO444,PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343, PRO1379,PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571, PRO1572,PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381, PRO4407,PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017, PRO7174,PRO9744, PRO9821, PRO9852, PRO9873, PRO10196, PRO34778, PRO20233,PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161 polypeptides arecontemplated.

The invention also provides fragments of a PRO226, PRO257, PRO268,PRO290, PRO36006, PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071,PRO1125, PRO1134, PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387,PRO1419, PRO1433, PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904,PRO35193, PRO4341, PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985,PRO4989, PRO5737, PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821,PRO9852, PRO9873, PRO10196, PRO34778, PRO20233, PRO21956, PRO57290,PRO38465, PRO38683 or PRO85161 polypeptide coding sequence, or thecomplement thereof, that may find use as, for example, hybridizationprobes, for encoding fragments of a PRO226, PRO257, PRO268, PRO290,PRO36006, PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125,PRO1134, PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419,PRO1433, PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193,PRO4341, PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989,PRO5737, PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852,PRO9873, PRO10196, PRO34778, PRO20233, PRO21956, PRO57290, PRO38465,PRO38683 or PRO85161 polypeptide that may optionally encode apolypeptide comprising a binding site for an anti-PRO226, anti-PRO257,anti-PRO268, anti-PRO290, anti-PRO36006, anti-PRO363, anti-PRO365,anti-PRO382, anti-PRO444, anti-PRO705, anti-PRO1071, anti-PRO1125,anti-PRO1134, anti-PRO1155, anti-PRO1281, anti-PRO1343, anti-PRO1379,anti-PRO1380, anti-PRO1387, anti-PRO1419, anti-PRO1433, anti-PRO1474,anti-PRO1550, anti-PRO1571, anti-PRO1572, anti-PRO1759, anti-PRO1904,anti-PRO35193, anti-PRO4341, anti-PRO4348, anti-PRO4369, anti-PRO4381,anti-PRO4407, anti-PRO4425, anti-PRO4985, anti-PRO4989, anti-PRO5737,anti-PRO5800, anti-PRO5993, anti-PRO6017, anti-PRO7174, anti-PRO9744,anti-PRO9821, anti-PRO9852, anti-PRO9873, anti-PRO10196, anti-PRO34778,anti-PRO20233, anti-PRO21956, anti-PRO57290, anti-PRO38465,anti-PRO38683 or anti-PRO85161 antibody or as antisense oligonucleotideprobes. Such nucleic acid fragments usually are or are at least about 10nucleotides in length, alternatively are or are at least about 15nucleotides in length, alternatively are or are at least about 20nucleotides in length, alternatively are or are at least about 30nucleotides in length, alternatively are or are at least about 40nucleotides in length, alternatively are or are at least about 50nucleotides in length, alternatively are or are at least about 60nucleotides in length, alternatively are or are at least about 70nucleotides in length, alternatively are or are at least about 80nucleotides in length, alternatively are or are at least about 90nucleotides in length, alternatively are or are at least about 100nucleotides in length, alternatively are or are at least about 110nucleotides in length, alternatively are or are at least about 120nucleotides in length, alternatively are or are at least about 130nucleotides in length, alternatively are or are at least about 140nucleotides in length, alternatively are or are at least about 150nucleotides in length, alternatively are or are at least about 160nucleotides in length, alternatively are or are at least about 170nucleotides in length, alternatively are or are at least about 180nucleotides in length, alternatively are or are at least about 190nucleotides in length, alternatively are or are at least about 200nucleotides in length, alternatively are or are at least about 250nucleotides in length, alternatively are or are at least about 300nucleotides in length, alternatively are or are at least about 350nucleotides in length, alternatively are or are at least about 400nucleotides in length, alternatively are or are at least about 450nucleotides in length, alternatively are or are at least about 500nucleotides in length, alternatively are or are at least about 600nucleotides in length, alternatively are or are at least about 700nucleotides in length, alternatively are or are at least about 800nucleotides in length, alternatively are or are at least about 900nucleotides in length and alternatively are or are at least about 1000nucleotides in length, wherein in this context the term “about” meansthe referenced nucleotide sequence length plus or minus 10% of thatreferenced length. It is noted that novel fragments of a PRO226, PRO257,PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382, PRO444, PRO705,PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343, PRO1379, PRO1380,PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571, PRO1572, PRO1759,PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381, PRO4407, PRO4425,PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017, PRO7174, PRO9744,PRO9821, PRO9852, PRO9873, PRO10196, PRO34778, PRO20233, PRO21956,PRO57290, PRO38465, PRO38683 or PRO85161 polypeptide-encoding nucleotidesequence may be determined in a routine manner by aligning the PRO226,PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382, PRO444,PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343, PRO1379,PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571, PRO1572,PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381, PRO4407,PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017, PRO7174,PRO9744, PRO9821, PRO9852, PRO9873, PRO10196, PRO34778, PRO20233,PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161 polypeptide-encodingnucleotide sequence with other known nucleotide sequences using any of anumber of well known sequence alignment programs and determining whichPRO226, PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382,PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343,PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571,PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381,PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017,PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196, PRO34778,PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161polypeptide-encoding nucleotide sequence fragment(s) are novel. All ofsuch PRO226, PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382,PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343,PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571,PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381,PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017,PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196, PRO34778,PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161polypeptide-encoding nucleotide sequences are contemplated herein. Alsocontemplated are the PRO226, PRO257, PRO268, PRO290, PRO36006, PRO363,PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155,PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474,PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348,PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800,PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196,PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161polypeptide fragments encoded by these nucleotide molecule fragments,preferably those PRO226, PRO257, PRO268, PRO290, PRO36006, PRO363,PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155,PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474,PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348,PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800,PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196,PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161polypeptide fragments that comprise a binding site for an anti-PRO226,anti-PRO257, anti-PRO268, anti-PRO290, anti-PRO36006, anti-PRO363,anti-PRO365, anti-PRO382, anti-PRO444, anti-PRO705, anti-PRO1071,anti-PRO1125, anti-PRO1134, anti-PRO1155, anti-PRO1281, anti-PRO1343,anti-PRO1379, anti-PRO1380, anti-PRO1387, anti-PRO1419, anti-PRO1433,anti-PRO1474, anti-PRO1550, anti-PRO1571, anti-PRO1572, anti-PRO1759,anti-PRO1904, anti-PRO35193, anti-PRO4341, anti-PRO4348, anti-PRO4369,anti-PRO4381, anti-PRO4407, anti-PRO4425, anti-PRO4985, anti-PRO4989,anti-PRO5737, anti-PRO5800, anti-PRO5993, anti-PRO6017, anti-PRO7174,anti-PRO9744, anti-PRO9821, anti-PRO9852, anti-PRO9873, anti-PRO10196,anti-PRO34778, anti-PRO20233, anti-PRO21956, anti-PRO57290,anti-PRO38465, anti-PRO38683 or anti-PRO85161 antibody.

The invention provides isolated PRO226, PRO257, PRO268, PRO290,PRO36006, PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125,PRO1134, PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419,PRO1433, PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193,PRO4341, PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989,PRO5737, PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852,PRO9873, PRO10196, PRO34778, PRO20233, PRO21956, PRO57290, PRO38465,PRO38683 or PRO85161 polypeptides encoded by any of the isolated nucleicacid sequences hereinabove identified.

In a certain aspect, the invention concerns an isolated PRO226, PRO257,PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382, PRO444, PRO705,PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343, PRO1379, PRO1380,PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571, PRO1572, PRO1759,PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381, PRO4407, PRO4425,PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017, PRO7174, PRO9744,PRO9821, PRO9852, PRO9873, PRO10196, PRO34778, PRO20233, PRO21956,PRO57290, PRO38465, PRO38683 or PRO85161 polypeptide, comprising anamino acid sequence having at least about 80% amino acid sequenceidentity, alternatively at least about 81% amino acid sequence identity,alternatively at least about 82% amino acid sequence identity,alternatively at least about 83% amino acid sequence identity,alternatively at least about 84% amino acid sequence identity,alternatively at least about 85% amino acid sequence identity,alternatively at least about 86% amino acid sequence identity,alternatively at least about 87% amino acid sequence identity,alternatively at least about 88% amino acid sequence identity,alternatively at least about 89% amino acid sequence identity,alternatively at least about 90% amino acid sequence identity,alternatively at least about 91% amino acid sequence identity,alternatively at least about 92% amino acid sequence identity,alternatively at least about 93% amino acid sequence identity,alternatively at least about 94% amino acid sequence identity,alternatively at least about 95% amino acid sequence identity,alternatively at least about 96% amino acid sequence identity,alternatively at least about 97% amino acid sequence identity,alternatively at least about 98% amino acid sequence identity andalternatively at least about 99% amino acid sequence identity to aPRO226, PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382,PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343,PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571,PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381,PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017,PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196, PRO34778,PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161 polypeptidehaving a full-length amino acid sequence as disclosed herein, an aminoacid sequence lacking the signal peptide as disclosed herein, anextracellular domain of a transmembrane protein, with or without thesignal peptide, as disclosed herein or any other specifically definedfragment of the full-length amino acid sequence as disclosed herein.

In a further aspect, the invention concerns an isolated PRO226, PRO257,PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382, PRO444, PRO705,PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343, PRO1379, PRO1380,PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571, PRO1572, PRO1759,PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381, PRO4407, PRO4425,PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017, PRO7174, PRO9744,PRO9821, PRO9852, PRO9873, PRO10196, PRO34778, PRO20233, PRO21956,PRO57290, PRO38465, PRO38683 or PRO85161 polypeptide comprising an aminoacid sequence having at least about 80% amino acid sequence identity,alternatively at least about 81% amino acid sequence identity,alternatively at least about 82% amino acid sequence identity,alternatively at least about 83% amino acid sequence identity,alternatively at least about 84% amino acid sequence identity,alternatively at least about 85% amino acid sequence identity,alternatively at least about 86% amino acid sequence identity,alternatively at least about 87% amino acid sequence identity,alternatively at least about 88% amino acid sequence identity,alternatively at least about 89% amino acid sequence identity,alternatively at least about 90% amino acid sequence identity,alternatively at least about 91% amino acid sequence identity,alternatively at least about 92% amino acid sequence identity,alternatively at least about 93% amino acid sequence identity,alternatively at least about 94% amino acid sequence identity,alternatively at least about 95% amino acid sequence identity,alternatively at least about 96% amino acid sequence identity,alternatively at least about 97% amino acid sequence identity,alternatively at least about 98% amino acid sequence identity andalternatively at least about 99% amino acid sequence identity to anamino acid sequence encoded by any of the human protein cDNAs depositedwith the ATCC as disclosed herein.

In one aspect, the invention concerns PRO226, PRO257, PRO268, PRO290,PRO36006, PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125,PRO1134, PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419,PRO1433, PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193,PRO4341, PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989,PRO5737, PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852,PRO9873, PRO10196, PRO34778, PRO20233, PRO21956, PRO57290, PRO38465,PRO38683 or PRO85161 variant polypeptides which are or are at leastabout 10 amino acids in length, alternatively are or are at least about20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170,180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310,320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450,460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590,600 amino acids in length, or more. Optionally, PRO226, PRO257, PRO268,PRO290, PRO36006, PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071,PRO1125, PRO1134, PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387,PRO1419, PRO1433, PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904,PRO35193, PRO4341, PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985,PRO4989, PRO5737, PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821,PRO9852, PRO9873, PRO10196, PRO34778, PRO20233, PRO21956, PRO57290,PRO38465, PRO38683 or PRO85161 variant polypeptides will have or have nomore than one conservative amino acid substitution as compared to thenative PRO226, PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382,PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343,PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571,PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381,PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017,PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196, PRO34778,PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161 polypeptidesequence, alternatively will have or will have no more than 2, 3, 4, 5,6, 7, 8, 9, or 10 conservative amino acid substitution as compared tothe native PRO226, PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365,PRO382, PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281,PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550,PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369,PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993,PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196,PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161polypeptide sequence.

In a specific aspect, the invention provides an isolated PRO226, PRO257,PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382, PRO444, PRO705,PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343, PRO1379, PRO1380,PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571, PRO1572, PRO1759,PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381, PRO4407, PRO4425,PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017, PRO7174, PRO9744,PRO9821, PRO9852, PRO9873, PRO10196, PRO34778, PRO20233, PRO21956,PRO57290, PRO38465, PRO38683 or PRO85161 polypeptide without theN-terminal signal sequence and/or the initiating methionine and isencoded by a nucleotide sequence that encodes such an amino acidsequence as hereinbefore described. Processes for producing the same arealso herein described, wherein those processes comprise culturing a hostcell comprising a vector which comprises the appropriate encodingnucleic acid molecule under conditions suitable for expression of thePRO226, PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382,PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343,PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571,PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381,PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017,PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196, PRO34778,PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161 polypeptideand recovering the PRO226, PRO257, PRO268, PRO290, PRO36006, PRO363,PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155,PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474,PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348,PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800,PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196,PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161polypeptide from the cell culture.

Another aspect the invention provides an isolated PRO226, PRO257,PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382, PRO444, PRO705,PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343, PRO1379, PRO1380,PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571, PRO1572, PRO1759,PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381, PRO4407, PRO4425,PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017, PRO7174, PRO9744,PRO9821, PRO9852, PRO9873, PRO10196, PRO34778, PRO20233, PRO21956,PRO57290, PRO38465, PRO38683 or PRO85161 polypeptide which is eithertransmembrane domain-deleted or transmembrane domain-inactivated.Processes for producing the same are also herein described, whereinthose processes comprise culturing a host cell comprising a vector whichcomprises the appropriate encoding nucleic acid molecule underconditions suitable for expression of the PRO226, PRO257, PRO268,PRO290, PRO36006, PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071,PRO1125, PRO1134, PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387,PRO1419, PRO1433, PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904,PRO35193, PRO4341, PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985,PRO4989, PRO5737, PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821,PRO9852, PRO9873, PRO10196, PRO34778, PRO20233, PRO21956, PRO57290,PRO38465, PRO38683 or PRO85161 polypeptide and recovering the PRO226,PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382, PRO444,PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343, PRO1379,PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571, PRO1572,PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381, PRO4407,PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017, PRO7174,PRO9744, PRO9821, PRO9852, PRO9873, PRO10196, PRO34778, PRO20233,PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161 polypeptide from thecell culture.

The invention provides agonists and antagonists of a native PRO226,PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382, PRO444,PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343, PRO1379,PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571, PRO1572,PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381, PRO4407,PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017, PRO7174,PRO9744, PRO9821, PRO9852, PRO9873, PRO10196, PRO34778, PRO20233,PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161 polypeptide asdefined herein. In particular, the agonist or antagonist is ananti-PRO226, anti-PRO257, anti-PRO268, anti-PRO290, anti-PRO36006,anti-PRO363, anti-PRO365, anti-PRO382, anti-PRO444, anti-PRO705,anti-PRO1071, anti-PRO1125, anti-PRO1134, anti-PRO1155, anti-PRO1281,anti-PRO1343, anti-PRO1379, anti-PRO1380, anti-PRO1387, anti-PRO1419,anti-PRO1433, anti-PRO1474, anti-PRO1550, anti-PRO1571, anti-PRO1572,anti-PRO1759, anti-PRO1904, anti-PRO35193, anti-PRO4341, anti-PRO4348,anti-PRO4369, anti-PRO4381, anti-PRO4407, anti-PRO4425, anti-PRO4985,anti-PRO4989, anti-PRO5737, anti-PRO5800, anti-PRO5993, anti-PRO6017,anti-PRO7174, anti-PRO9744, anti-PRO9821, anti-PRO9852, anti-PRO9873,anti-PRO10196, anti-PRO34778, anti-PRO20233, anti-PRO21956,anti-PRO57290, anti-PRO38465, anti-PRO38683 or anti-PRO85161 antibody ora small molecule.

The invention provides a method of identifying agonists or antagoniststo a PRO226, PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382,PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343,PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571,PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381,PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017,PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196, PRO34778,PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161 polypeptidewhich comprise contacting the PRO226, PRO257, PRO268, PRO290, PRO36006,PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125, PRO1134,PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433,PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341,PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737,PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873,PRO10196, PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 orPRO85161 polypeptide with a candidate molecule and monitoring abiological activity mediated by said PRO226, PRO257, PRO268, PRO290,PRO36006, PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125,PRO1134, PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419,PRO1433, PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193,PRO4341, PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989,PRO5737, PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852,PRO9873, PRO10196, PRO34778, PRO20233, PRO21956, PRO57290, PRO38465,PRO38683 or PRO85161 polypeptide. Preferably, the PRO226, PRO257,PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382, PRO444, PRO705,PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343, PRO1379, PRO1380,PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571, PRO1572, PRO1759,PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381, PRO4407, PRO4425,PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017, PRO7174, PRO9744,PRO9821, PRO9852, PRO9873, PRO10196, PRO34778, PRO20233, PRO21956,PRO57290, PRO38465, PRO38683 or PRO85161 polypeptide is a native PRO226,PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382, PRO444,PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343, PRO1379,PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571, PRO1572,PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381, PRO4407,PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017, PRO7174,PRO9744, PRO9821, PRO9852, PRO9873, PRO10196, PRO34778, PRO20233,PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161 polypeptide.

The invention provides a composition of matter comprising a PRO226,PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382, PRO444,PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343, PRO1379,PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571, PRO1572,PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381, PRO4407,PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017, PRO7174,PRO9744, PRO9821, PRO9852, PRO9873, PRO10196, PRO34778, PRO20233,PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161 polypeptide, or anagonist or antagonist of a PRO226, PRO257, PRO268, PRO290, PRO36006,PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125, PRO1134,PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433,PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341,PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737,PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873,PRO10196, PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 orPRO85161 polypeptide as herein described, or an anti-PRO226,anti-PRO257, anti-PRO268, anti-PRO290, anti-PRO36006, anti-PRO363,anti-PRO365, anti-PRO382, anti-PRO444, anti-PRO705, anti-PRO1071,anti-PRO1125, anti-PRO1134, anti-PRO1155, anti-PRO1281, anti-PRO1343,anti-PRO1379, anti-PRO1380, anti-PRO1387, anti-PRO1419, anti-PRO1433,anti-PRO1474, anti-PRO1550, anti-PRO1571, anti-PRO1572, anti-PRO1759,anti-PRO1904, anti-PRO35193, anti-PRO4341, anti-PRO4348, anti-PRO4369,anti-PRO4381, anti-PRO4407, anti-PRO4425, anti-PRO4985, anti-PRO4989,anti-PRO5737, anti-PRO5800, anti-PRO5993, anti-PRO6017, anti-PRO7174,anti-PRO9744, anti-PRO9821, anti-PRO9852, anti-PRO9873, anti-PRO10196,anti-PRO34778, anti-PRO20233, anti-PRO21956, anti-PRO57290,anti-PRO38465, anti-PRO38683 or anti-PRO85161 antibody, in combinationwith a carrier. Optionally, the carrier is a pharmaceutically acceptablecarrier.

The invention provides the use of a PRO226, PRO257, PRO268, PRO290,PRO36006, PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125,PRO1134, PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419,PRO1433, PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193,PRO4341, PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989,PRO5737, PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852,PRO9873, PRO10196, PRO34778, PRO20233, PRO21956, PRO57290, PRO38465,PRO38683 or PRO85161 polypeptide, or an agonist or antagonist thereof ashereinbefore described, or an anti-PRO226, anti-PRO257, anti-PRO268,anti-PRO290, anti-PRO36006, anti-PRO363, anti-PRO365, anti-PRO382,anti-PRO444, anti-PRO705, anti-PRO1071, anti-PRO1125, anti-PRO1134,anti-PRO1155, anti-PRO1281, anti-PRO1343, anti-PRO1379, anti-PRO1380,anti-PRO1387, anti-PRO1419, anti-PRO1433, anti-PRO1474, anti-PRO1550,anti-PRO1571, anti-PRO1572, anti-PRO1759, anti-PRO1904, anti-PRO35193,anti-PRO4341, anti-PRO4348, anti-PRO4369, anti-PRO4381, anti-PRO4407,anti-PRO4425, anti-PRO4985, anti-PRO4989, anti-PRO5737, anti-PRO5800,anti-PRO5993, anti-PRO6017, anti-PRO7174, anti-PRO9744, anti-PRO9821,anti-PRO9852, anti-PRO9873, anti-PRO10196, anti-PRO34778, anti-PRO20233,ant, anti-PRO21956 anti-PRO57290, anti-PRO38465, anti-PRO38683 oranti-PRO85161 antibody, for the preparation of a medicament useful inthe treatment of a condition which is responsive to the anti-PRO226,anti-PRO257, anti-PRO268, anti-PRO290, anti-PRO36006, anti-PRO363,anti-PRO365, anti-PRO382, anti-PRO444, anti-PRO705, anti-PRO1071,anti-PRO1125, anti-PRO1134, anti-PRO1155, anti-PRO1281, anti-PRO1343,anti-PRO1379, anti-PRO1380, anti-PRO1387, anti-PRO1419, anti-PRO1433,anti-PRO1474, anti-PRO1550, anti-PRO1571, anti-PRO1572, anti-PRO1759,anti-PRO1904, anti-PRO35193, anti-PRO4341, anti-PRO4348, anti-PRO4369,anti-PRO4381, anti-PRO4407, anti-PRO4425, anti-PRO4985, anti-PRO4989,anti-PRO5737, anti-PRO5800, anti-PRO5993, anti-PRO6017, anti-PRO7174,anti-PRO9744, anti-PRO9821, anti-PRO9852, anti-PRO9873, anti-PRO10196,anti-PRO34778, anti-PRO20233, anti-PRO21956, anti-PRO57290,anti-PRO38465, anti-PRO38683 or anti-PRO85161 antibody.

The invention provides vectors comprising DNA encoding any of the hereindescribed polypeptides. Host cell comprising any such vector are alsoprovided. By way of example, the host cells may be CHO cells, E. coli,or yeast. A process for producing any of the herein describedpolypeptides is further provided and comprises culturing host cellsunder conditions suitable for expression of the desired polypeptide andrecovering the desired polypeptide from the cell culture.

The invention provides chimeric molecules comprising any of the hereindescribed polypeptides fused to a heterologous polypeptide or amino acidsequence. Example of such chimeric molecules comprise any of the hereindescribed polypeptides fused to an epitope tag sequence or a Fc regionof an immunoglobulin.

The invention provides an antibody which binds, preferably specifically,to any of the above or below described polypeptides. Optionally, theantibody is a monoclonal antibody, humanized antibody, antibody fragmentor single-chain antibody.

The invention provides oligonucleotide probes which may be useful forisolating genomic and cDNA nucleotide sequences, measuring or detectingexpression of an associated gene or as antisense probes, wherein thoseprobes may be derived from any of the above or below describednucleotide sequences. Preferred probe lengths are described above.

The invention also provides a method of identifying a phenotypeassociated with a disruption of a gene which encodes for a PRO226,PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382, PRO444,PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343, PRO1379,PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571, PRO1572,PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381, PRO4407,PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017, PRO7174,PRO9744, PRO9821, PRO9852, PRO9873, PRO10196, PRO34778, PRO20233,PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161 polypeptide, themethod comprising:

(a) providing a non-human transgenic animal whose genome comprises adisruption of the gene which encodes for a PRO226, PRO257, PRO268,PRO290, PRO36006, PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071,PRO1125, PRO1134, PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387,PRO1419, PRO1433, PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904,PRO35193, PRO4341, PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985,PRO4989, PRO5737, PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821,PRO9852, PRO9873, PRO10196, PRO34778, PRO20233, PRO21956, PRO57290,PRO38465, PRO38683 or PRO85161 polypeptide;

(b) measuring a physiological characteristic of the non-human transgenicanimal; and

(c) comparing the measured physiological characteristic with that of agender matched wild-type animal, wherein the physiologicalcharacteristic of the non-human transgenic animal that differs from thephysiological characteristic of the wild-type animal is identified as aphenotype resulting from the gene disruption in the non-human transgenicanimal. In one aspect, the non-human transgenic animal is a mammal. Inanother aspect, the mammal is a rodent. In still another aspect, themammal is a rat or a mouse. In one aspect, the non-human transgenicanimal is heterozygous for the disruption of a gene which encodes for aPRO226, PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382,PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343,PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571,PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381,PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017,PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196, PRO34778,PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161polypeptide. In another aspect, the phenotype exhibited by the non-humantransgenic animal as compared with gender matched wild-type littermatesis at least one of the following: a neurological disorder; acardiovascular, endothelial or angiogenic disorder; an eye abnormality;an immunological disorder; an oncological disorder; a bone metabolicabnormality or disorder; a lipid metabolic disorder; or a developmentalabnormality.

In yet another aspect, the neurological disorder is an increasedanxiety-like response during open field activity testing. In yet anotheraspect, the neurological disorder is a decreased anxiety-like responseduring open field activity testing. In yet another aspect, theneurological disorder is an abnormal circadian rhythm during home-cageactivity testing. In yet another aspect, the neurological disorder is anenhanced motor coordination during inverted screen testing. In yetanother aspect, the neurological disorder is impaired motor coordinationduring inverted screen testing. In yet another aspect, the neurologicaldisorder includes depression, generalized anxiety disorders, attentiondeficit disorder, sleep disorder, hyperactivity disorder, obsessivecompulsive disorder, schizophrenia, cognitive disorders, hyperalgesiaand sensory disorders. Such neurological disorders include the categorydefined as “anxiety disorders” which include but are not limited to:mild to moderate anxiety, anxiety disorder due to a general medicalcondition, anxiety disorder not otherwise specified, generalized anxietydisorder, panic attack, panic disorder with agoraphobia, panic disorderwithout agoraphobia, posttraumatic stress disorder, social phobia,social anxiety, autism, specific phobia, substance-induced anxietydisorder, acute alcohol withdrawal, obsessive compulsive disorder,agoraphobia, monopolar disorders, bipolar disorder I or II, bipolardisorder not otherwise specified, cyclothymic disorder, depressivedisorder, major depressive disorder, mood disorder, substance-inducedmood disorder, enhancement of cognitive function, loss of cognitivefunction associated with but not limited to Alzheimer's disease, stroke,or traumatic injury to the brain, seizures resulting from disease orinjury including but not limited to epilepsy, learningdisorders/disabilities, cerebral palsy. In addition, anxiety disordersmay apply to personality disorders including but not limited to thefollowing types: paranoid, antisocial, avoidant behavior, borderlinepersonality disorders, dependent, histrionic, narcissistic,obsessive-compulsive, schizoid, and schizotypal.

In another aspect, the eye abnormality is a retinal abnormality. Instill another aspect, the eye abnormality is consistent with visionproblems or blindness. In yet another aspect, the retinal abnormality isconsistent with retinitis pigmentosa or is characterized by retinaldegeneration or retinal dysplasia.

In still another aspect, the retinal abnormalities are consistent withretinal dysplasia, various retinopathies, including retinopathy ofprematurity, retrolental fibroplasia, neovascular glaucoma, age-relatedmacular degeneration, diabetic macular edema, cornealneovascularization, corneal graft neovascularization, corneal graftrejection, retinal/choroidal neovascularization, neovascularization ofthe angle (rubeosis), ocular neovascular disease, vascular restenosis,arteriovenous malformations (AVM), meningioma, hemangioma, angiofibroma,thyroid hyperplasias (including Grave's disease), corneal and othertissue transplantation, retinal artery obstruction or occlusion; retinaldegeneration causing secondary atrophy of the retinal vasculature,retinitis pigmentosa, macular dystrophies, Stargardt's disease,congenital stationary night blindness, choroideremia, gyrate atrophy,Leber's congenital amaurosis, retinoschisis disorders, Wagner'ssyndrome, Usher syndromes, Zellweger syndrome, Saldino-Mainzer syndrome,Senior-Loken syndrome, Bardet-Biedl syndrome, Alport's syndrome,Alstrom's syndrome, Cockayne's syndrome, dysplaisa spondyloepiphysariacongentia, Flynn-Aird syndrome, Friedreich ataxia, Hallgren syndrome,Marshall syndrome, Albers-Schnoberg disease, Refsum's disease,Kearns-Sayre syndrome, Waardenburg's syndrome, Alagile syndrome,myotonic dystrophy, olivopontocerebellar atrophy, Pierre-Mariedunsdrome, Stickler syndrome, carotinemeia, cystinosis, Wolframsyndrome, Bassen-Kornzweig syndrome, abetalipoproteinemia, incontinentiapigmenti, Batten's disease, mucopolysaccharidoses, homocystinuria, ormannosidosis.

In still another aspect, the eye abnormality is a cataract. In still yetanother aspect, the cataract is a systemic disease such as human Down'ssyndrome, Hallerman-Streiff syndrome, Lowe syndrome, galactosemia,Marfan syndrome, Trismoy 13-15, Alport syndrome, myotonic dystrophy,Fabry disease, hypoparathroidism or Conradi syndrome.

In still another aspect, the developmental abnormality comprisesembryonic lethality or reduced viability.

In still yet another aspect, the cardiovascular, endothelial orangiogenic disorders are arterial diseases, such as diabetes mellitus;papilledema; optic atrophy; atherosclerosis; angina; myocardialinfarctions such as acute myocardial infarctions, cardiac hypertrophy,and heart failure such as congestive heart failure; hypertension;inflammatory vasculitides; Reynaud's disease and Reynaud's phenomenon;aneurysms and arterial restenosis; venous and lymphatic disorders suchas thrombophlebitis, lymphangitis, and lymphedema; peripheral vasculardisease; cancer such as vascular tumors, e.g., hemangioma (capillary andcavernous), glomus tumors, telangiectasia, bacillary angiomatosis,hemangioendothelioma, angiosarcoma, haemangiopericytoma, Kaposi'ssarcoma, lymphangioma, and lymphangiosarcoma; tumor angiogenesis; traumasuch as wounds, burns, and other injured tissue, implant fixation,scarring; ischemia reperfusion injury; rheumatoid arthritis;cerebrovascular disease; renal diseases such as acute renal failure, orosteoporosis.

In still another aspect, the immunological disorders are consistent withsystemic lupus erythematosis; rheumatoid arthritis; juvenile chronicarthritis; spondyloarthropathies; systemic sclerosis (scleroderma);idiopathic inflammatory myopathies (dermatomyositis, polymyositis);Sjögren's syndrome; systemic vasculitis; sarcoidosis; autoimmunehemolytic anemia (immune pancytopenia, paroxysmal nocturnalhemoglobinuria); autoimmune thrombocytopenia (idiopathicthrombocytopenic purpura, immune-mediated thrombocytopenia); thyroiditis(Grave's disease, Hashimoto's thyroiditis, juvenile lymphocyticthyroiditis, atrophic thyroiditis); diabetes mellitus; immune-mediatedrenal disease (glomerulonephritis, tubulointerstitial nephritis);demyelinating diseases of the central and peripheral nervous systemssuch as multiple sclerosis, idiopathic demyelinating polyneuropathy orGuillain-Barré syndrome, and chronic inflammatory demyelinatingpolyneuropathy; hepatobiliary diseases such as infectious hepatitis(hepatitis A, B, C, D, E and other non-hepatotropic viruses), autoimmunechronic active hepatitis, primary biliary cirrhosis, granulomatoushepatitis, and sclerosing cholangitis; inflammatory bowel disease(ulcerative colitis: Crohn's disease); gluten-sensitive enteropathy, andWhipple's disease; autoimmune or immune-mediated skin diseases includingbullous skin diseases, erythema multiforme and contact dermatitis,psoriasis; allergic diseases such as asthma, allergic rhinitis, atopicdermatitis, food hypersensitivity and urticaria; immunologic diseases ofthe lung such as eosinophilic pneumonia, idiopathic pulmonary fibrosisand hypersensitivity pneumonitis; or transplantation associated diseasesincluding graft rejection and graft-versus-host disease.

In still another aspect, the bone metabolic abnormality or disorder isarthritis, osteoporosis, osteopenia or osteopetrosis.

In another aspect, the non-human transgenic animal exhibits at least oneof the following physiological characteristics compared with gendermatched wild-type littermates: increased anxiety-like response duringopen field testing; decreased anxiety during open field testing;decreased locomotor activity during open field testing; abnormalcircadian rhythm during home-cage activity testing (low activity duringthe light phase); abnormal circadian rhythm during home-cage activitytesting including decreased ambulatory counts; hypoactivity with nocircadian rhythm; abnormal circadian rhythm during home-cage activitytesting including increased ambulatory counts; increased stress inducedhyperthermia; decreased stress induced hyperthermia; impaired motorcoordination during inverted screen testing; increased immobility intail suspension (increased depressive-like response); increaseddepressive-like response during tail suspension testing; increasedimmobility or decreased depressive-like response during tail suspensiontesting; decreased startle response during prepulse inhibition testing;no startle response indicating deafness; or impaired hearing; decreasedprepulse inhibition with impaired sensorimotor gating/attention;decreased responsiveness in hot plate testing; decreased latency torespond in hot plate testing; opthamological abnormalities; increasedmean artery-to-vein ratio; resistance to pupil dilating drugcyclopentolate hydrochloride; squinty eyes; squint eyes with whitespots; cataracts; retinal degeneration; impaired vision; decreased basalbody temperature; decreased heart rate; increased mean systolic bloodpressure; increased insulin sensitivity; increased mean fasting serumglucose levels; decreased mean serum glucose levels; increased meanserum cholesterol levels; decreased mean serum cholesterol levels;increased mean serum triglyceride levels; decreased mean serumtriglyceride levels; enhanced glucose tolerance; impaired glucosetolerance; decreased mean serum insulin levels; increased mean serumcalcium; increased urobilinogen, notable lipemia; increased albumin,alanine amino transferase, phosphorus and potassium levels; increasedmean serum alkaline phosphatase levels; increased blood urea nitrogen;increased percentage of granulocyte; increased total white blood cell(WBC) count; increased mean absolute neutrophil count; neutropenia;increased absolute lymphocyte count; increased absolute monocyte count;increased monocytes and DC in spleen (CD11b+, CD11b+c+); increased meanplatelet count; increased natural killer (NK) cells in lymph node;decreased neutrophil count; decreased natural killer (NK) cells;decreased mean red blood cell (RBC) count, hemoglobin concentration, andhematocrit; increased mean red cell distribution width; decreased meancorpuscular volume and mean corpuscular hemoglobin; decreased meanplatelet count and increased platelet volume; increase B cell number inlymph node; increase in B cell subtypes in Peyer's patches; increasedpercentage of B cells in lymph node; increase CD25+ cells; increasedthymic DN, decreased DP T cells; increased CD19+ cells in lymph node;increased CD117 in bone marrow cells; increased mean percentage of CD4cells; increased CD8 cells and decrease in B cells; increased percentageCD11b+ cells in peritoneal lavage; increased percentage of B220+CD11bLow CD23− cells; increased percentages of B220− CD11 Low and CD11b−cells in peritoneal lavage; increased percentage of B220−CD11bHi cellsin peritoneal lavage; decreased percentage of B220+ CD11b− CD23+ cellsin peritoneal lavage; increased percentage of B220− CD43 Hi cells inbone marrow; increased CD11b+ CD11c− cells in spleen; increase inCD62hi, CD44int subsets of CD4 and CD8 cells; increase in peritonealCD117 cells; increase TcRbeta/CD38 cells in Peyer's patches; increasedpercentage of TcRbeta+ cells in thymus; increased percentages of CD11b+CD11c+ in lymph node; decreased percentage of B220+ Hi CD23+ cells inperitoneal lavage; decreased percentage of B220+ Med CD23− cells inperitoneal lavage; decreased percentages of CD62L Hi CD44 Dim CD4+ andCD8+ cells in spleen; decreased percentage of B220−CD11b Hi cells;decreased mean percentages of CD4 and CD8 cells in lymph node andspleen; increased memory T cells [increased CD62L lo CD44hi]; decreasedT cell:B cell ratio; decreased naive T cells; decreased CD117 cells inperitoneal lavage; decreased mean percentage of CD8 cells, increasedIgG1 response to ovalbumin challenge; increased IgG2a response toovalbumin challenge; increased mean serum IL-6 response to LPSchallenge; increased TNF alpha response to LPS challenge; increasedserum MCP-1 response to LPS challenge; increased mean serum IgM level;increased serum IgA; increase mean serum IgG1; increased mean serumIgG3; decreased serum IgG1 response to ovalbumin challenge; decreasedserum IgG2a response to ovalbumin challenge; decreased mean serum IgAlevel; decreased serum IgG2a level; decrease in serum IgG3 level;increased skin fibroblast proliferation rate; decreased skin fibroblastproliferation rate; increased mean percent of total body fat and totalfat mass; increased mean body weight; increased mean body length;increased total tissue mass (TTM); increased mean femoral midshaftcortical thickness and cross-sectional area; increased mean vertebraltrabecular bone volume, number and connectivity density; decreased meanpercent of total body fat and total fat mass; decreased mean bodyweight; decreased mean body length; decreased total tissue mass (TTM);decreased lean body mass (LBM); decreased femoral bone mineral density(BMD); decreased vertebral bone mineral density (BMD); decreased bonemineral density (BMD) in total body, femur and vertebrae; decreased bonemineral content (BMC); decreased bone mineral density index; decreasedvolumetric bone mineral density (vBMD); decreased mean femoral midshaftcortical thickness and cross-sectional area; decreased mean vertebraltrabecular bone volume, number and connectivity density; osteopetrosis;osteoporosis; chronic inflammation in various tissues; bilateralhydronephrosis (moderate to severe) and inflammation; “pear shapedabdomen”; bilaterally enlarged kidneys, suggesting polycystic kidneydisease; degeneration of the Organ of Corti; hepatocellular dysfunction;biliary obstruction; hepatosplenomegaly characterized by histiocyticinfiltrate; histiocytosis in the small intestine, lymph nodes andspleen; splenomegaly, lymphadenopathy and lymphadenopathy; hyperplasiaof adenoid and tonsils; mild-moderate extra medullary hematopoiesis;homozygous mice were small, dehydrated and exhibited decreasedsubcutaneous fat depots; lipopenia; ulcerous colitis; diffuse markeddegeneration of sensory cochlear hair cells in the inner ear,characterized by a complete loss of both inner and outer cochlear haircells on the basilar membrane; gastric mucosal hyperplasia and chronicinflammation; in creased stomach weight; defective spermatogenesis inthe testes; hypospermia and defective spermatozoa in the epididymus;male infertility; lysosomal storage disease; anemia; growth retardation;reduced viability; perinatal lethality with decreased lymphocytes andlipopenia; homozygous embryonic lethality; and heterozygous embryoniclethality.

The invention also provides an isolated cell derived from a non-humantransgenic animal whose genome comprises a disruption of the gene whichencodes for a PRO226, PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365,PRO382, PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281,PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550,PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369,PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993,PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196,PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161polypeptide. In one aspect, the isolated cell is a murine cell. In yetanother aspect, the murine cell is an embryonic stem cell. In stillanother aspect, the isolated cell is derived from a non-human transgenicanimal which exhibits at least one of the following phenotypes comparedwith gender matched wild-type littermates: a neurological disorder; acardiovascular, endothelial or angiogenic disorder; an eye abnormality;an immunological disorder; an oncological disorder; a bone metabolicabnormality or disorder; a lipid metabolic disorder; or a developmentalabnormality. The invention also provides a method of identifying anagent that modulates a phenotype associated with a disruption of a genewhich encodes for a PRO226, PRO257, PRO268, PRO290, PRO36006, PRO363,PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155,PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474,PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348,PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800,PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196,PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161polypeptide, the method comprising:

(a) providing a non-human transgenic animal whose genome comprises adisruption of the gene which encodes for the PRO226, PRO257, PRO268,PRO290, PRO36006, PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071,PRO1125, PRO1134, PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387,PRO1419, PRO1433, PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904,PRO35193, PRO4341, PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985,PRO4989, PRO5737, PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821,PRO9852, PRO9873, PRO10196, PRO34778, PRO20233, PRO21956, PRO57290,PRO38465, PRO38683 or PRO85161 polypeptide;

(b) measuring a physiological characteristic of the non-human transgenicanimal of (a);

(c) comparing the measured physiological characteristic of (b) with thatof a gender matched wild-type animal, wherein the physiologicalcharacteristic of the non-human transgenic animal that differs from thephysiological characteristic of the wild-type animal is identified as aphenotype resulting from the gene disruption in the non-human transgenicanimal;

(d) administering a test agent to the non-human transgenic animal of(a); and

(e) determining whether the test agent modulates the identifiedphenotype associated with gene disruption in the non-human transgenicanimal.

In one aspect, the phenotype associated with the gene disruptioncomprises a neurological disorder; a cardiovascular, endothelial orangiogenic disorder; an eye abnormality; an immunological disorder; anoncological disorder; a bone metabolic abnormality or disorder; a lipidmetabolic disorder; or a developmental abnormality.

In yet another aspect, the neurological disorder is an increasedanxiety-like response during open field activity testing. In yet anotheraspect, the neurological disorder is a decreased anxiety-like responseduring open field activity testing. In yet another aspect, theneurological disorder is an abnormal circadian rhythm during home-cageactivity testing. In yet another aspect, the neurological disorder is anenhanced motor coordination during inverted screen testing. In yetanother aspect, the neurological disorder is impaired motor coordinationduring inverted screen testing. In yet another aspect, the neurologicaldisorder includes depression, generalized anxiety disorders, attentiondeficit disorder, sleep disorder, hyperactivity disorder, obsessivecompulsive disorder, schizophrenia, cognitive disorders, hyperalgesiaand sensory disorders. Such neurological disorders include the categorydefined as “anxiety disorders” which include but are not limited to:mild to moderate anxiety, anxiety disorder due to a general medicalcondition, anxiety disorder not otherwise specified, generalized anxietydisorder, panic attack, panic disorder with agoraphobia, panic disorderwithout agoraphobia, posttraumatic stress disorder, social phobia,social anxiety, autism, specific phobia, substance-induced anxietydisorder, acute alcohol withdrawal, obsessive compulsive disorder,agoraphobia, monopolar disorders, bipolar disorder I or II, bipolardisorder not otherwise specified, cyclothymic disorder, depressivedisorder, major depressive disorder, mood disorder, substance-inducedmood disorder, enhancement of cognitive function, loss of cognitivefunction associated with but not limited to Alzheimer's disease, stroke,or traumatic injury to the brain, seizures resulting from disease orinjury including but not limited to epilepsy, learningdisorders/disabilities, cerebral palsy. In addition, anxiety disordersmay apply to personality disorders including but not limited to thefollowing types: paranoid, antisocial, avoidant behavior, borderlinepersonality disorders, dependent, histrionic, narcissistic,obsessive-compulsive, schizoid, and schizotypal.

In yet another aspect, the eye abnormality is a retinal abnormality. Instill another aspect, the eye abnormality is consistent with visionproblems or blindness. In yet another aspect, the retinal abnormality isconsistent with retinitis pigmentosa or is characterized by retinaldegeneration or retinal dysplasia.

In still another aspect, the retinal abnormalities are consistent withretinal dysplasia, various retinopathies, including retinopathy ofprematurity, retrolental fibroplasia, neovascular glaucoma, age-relatedmacular degeneration, diabetic macular edema, cornealneovascularization, corneal graft neovascularization, corneal graftrejection, retinal/choroidal neovascularization, neovascularization ofthe angle (rubeosis), ocular neovascular disease, vascular restenosis,arteriovenous malformations (AVM), meningioma, hemangioma, angiofibroma,thyroid hyperplasias (including Grave's disease), corneal and othertissue transplantation, retinal artery obstruction or occlusion; retinaldegeneration causing secondary atrophy of the retinal vasculature,retinitis pigmentosa, macular dystrophies, Stargardt's disease,congenital stationary night blindness, choroideremia, gyrate atrophy,Leber's congenital amaurosis, retinoschisis disorders, Wagner'ssyndrome, Usher syndromes, Zellweger syndrome, Saldino-Mainzer syndrome,Senior-Loken syndrome, Bardet-Biedl syndrome, Alport's syndrome,Alstrom's syndrome, Cockayne's syndrome, dysplaisa spondyloepiphysariacongentia, Flynn-Aird syndrome, Friedreich ataxia, Hallgren syndrome,Marshall syndrome, Albers-Schnoberg disease, Refsum's disease,Kearns-Sayre syndrome, Waardenburg's syndrome, Alagile syndrome,myotonic dystrophy, olivopontocerebellar atrophy, Pierre-Mariedunsdrome, Stickler syndrome, carotinemeia, cystinosis, Wolframsyndrome, Bassen-Kornzweig syndrome, abetalipoproteinemia, incontinentiapigmenti, Batten's disease, mucopolysaccharidoses, homocystinuria, ormannosidosis.

In still another aspect, the eye abnormality is a cataract. In still yetanother aspect, the cataract is a systemic disease such as human Down'ssyndrome, Hallerman-Streiff syndrome, Lowe syndrome, galactosemia,Marfan syndrome, Trismoy 13-15, Alport syndrome, myotonic dystrophy,Fabry disease, hypoparathroidism, or Conradi syndrome.

In still another aspect, the developmental abnormality comprisesembryonic lethality or reduced viability.

In still another aspect, the cardiovascular, endothelial or angiogenicdisorders are arterial diseases, such as diabetes mellitus; papilledema;optic atrophy; atherosclerosis; angina; myocardial infarctions such asacute myocardial infarctions, cardiac hypertrophy, and heart failuresuch as congestive heart failure; hypertension; inflammatoryvasculitides; Reynaud's disease and Reynaud's phenomenon; aneurysms andarterial restenosis; venous and lymphatic disorders such asthrombophlebitis, lymphangitis, and lymphedema; peripheral vasculardisease; cancer such as vascular tumors, e.g., hemangioma (capillary andcavernous), glomus tumors, telangiectasia, bacillary angiomatosis,hemangioendothelioma, angiosarcoma, haemangiopericytoma, Kaposi'ssarcoma, lymphangioma, and lymphangiosarcoma; tumor angiogenesis; traumasuch as wounds, burns, and other injured tissue, implant fixation,scarring; ischemia reperfusion injury; rheumatoid arthritis;cerebrovascular disease; renal diseases such as acute renal failure, orosteoporosis.

In still another aspect, the immunological disorders are consistent withsystemic lupus erythematosis; rheumatoid arthritis; juvenile chronicarthritis; spondyloarthropathies; systemic sclerosis (scleroderma);idiopathic inflammatory myopathies (dermatomyositis, polymyositis);Sjögren's syndrome; systemic vasculitis; sarcoidosis; autoimmunehemolytic anemia (immune pancytopenia, paroxysmal nocturnalhemoglobinuria); autoimmune thrombocytopenia (idiopathicthrombocytopenic purpura, immune-mediated thrombocytopenia); thyroiditis(Grave's disease, Hashimoto's thyroiditis, juvenile lymphocyticthyroiditis, atrophic thyroiditis); diabetes mellitus; immune-mediatedrenal disease (glomerulonephritis, tubulointerstitial nephritis);demyelinating diseases of the central and peripheral nervous systemssuch as multiple sclerosis, idiopathic demyelinating polyneuropathy orGuillain-Barré syndrome, and chronic inflammatory demyelinatingpolyneuropathy; hepatobiliary diseases such as infectious hepatitis(hepatitis A, B, C, D, E and other non-hepatotropic viruses), autoimmunechronic active hepatitis, primary biliary cirrhosis, granulomatoushepatitis, and sclerosing cholangitis; inflammatory bowel disease(ulcerative colitis: Crohn's disease); gluten-sensitive enteropathy, andWhipple's disease; autoimmune or immune-mediated skin diseases includingbullous skin diseases, erythema multiforme and contact dermatitis,psoriasis; allergic diseases such as asthma, allergic rhinitis, atopicdermatitis, food hypersensitivity and urticaria; immunologic diseases ofthe lung such as eosinophilic pneumonia, idiopathic pulmonary fibrosisand hypersensitivity pneumonitis; or transplantation associated diseasesincluding graft rejection and graft-versus-host disease.

In yet another aspect, the bone metabolic abnormality or disorder isarthritis, osteoporosis, osteopenia or osteopetrosis.

In another aspect, the non-human transgenic animal exhibits at least oneof the following physiological characteristics compared with gendermatched wild-type littermates: increased anxiety-like response duringopen field testing; decreased anxiety during open field testing;decreased locomotor activity during open field testing; abnormalcircadian rhythm during home-cage activity testing (low activity duringthe light phase); abnormal circadian rhythm during home-cage activitytesting including decreased ambulatory counts; hypoactivity with nocircadian rhythm; abnormal circadian rhythm during home-cage activitytesting including increased ambulatory counts; increased stress inducedhyperthermia; decreased stress induced hyperthermia; impaired motorcoordination during inverted screen testing; increased immobility intail suspension (increased depressive-like response); increaseddepressive-like response during tail suspension testing; increasedimmobility or decreased depressive-like response during tail suspensiontesting; decreased startle response during prepulse inhibition testing;no startle response indicating deafness; or impaired hearing; decreasedprepulse inhibition with impaired sensorimotor gating/attention;decreased responsiveness in hot plate testing; decreased latency torespond in hot plate testing; opthamological abnormalities; increasedmean artery-to-vein ratio; resistance to pupil dilating drugcyclopentolate hydrochloride; squinty eyes; squint eyes with whitespots; cataracts; retinal degeneration; impaired vision; decreased basalbody temperature; decreased heart rate; increased mean systolic bloodpressure; increased insulin sensitivity; increased mean fasting serumglucose levels; decreased mean serum glucose levels; increased meanserum cholesterol levels; decreased mean serum cholesterol levels;increased mean serum triglyceride levels; decreased mean serumtriglyceride levels; enhanced glucose tolerance; impaired glucosetolerance; decreased mean serum insulin levels; increased mean serumcalcium; increased urobilinogen, notable lipemia; increased albumin,alanine amino transferase, phosphorus and potassium levels; increasedmean serum alkaline phosphatase levels; increased blood urea nitrogen;increased percentage of granulocyte; increased total white blood cell(WBC) count; increased mean absolute neutrophil count; neutropenia;increased absolute lymphocyte count; increased absolute monocyte count;increased monocytes and DC in spleen (CD11b+, CD11b+c+); increased meanplatelet count; increased natural killer (NK) cells in lymph node;decreased neutrophil count; decreased natural killer (NK) cells;decreased mean red blood cell (RBC) count, hemoglobin concentration, andhematocrit; increased mean red cell distribution width; decreased meancorpuscular volume and mean corpuscular hemoglobin; decreased meanplatelet count and increased platelet volume; increase B cell number inlymph node; increase in B cell subtypes in Peyer's patches; increasedpercentage of B cells in lymph node; increase CD25+ cells; increasedthymic DN, decreased DP T cells; increased CD19+ cells in lymph node;increased CD117 in bone marrow cells; increased mean percentage of CD4cells; increased CD8 cells and decrease in B cells; increased percentageCD11b+ cells in peritoneal lavage; increased percentage of B220+ CD11bLow CD23− cells; increased percentages of B220− CD11 Low and CD11b−cells in peritoneal lavage; increased percentage of B220−CD11bhi cellsin peritoneal lavage; decreased percentage of B220+ CD11b− CD23+ cellsin peritoneal lavage; increased percentage of B220− CD43 Hi cells inbone marrow; increased CD11b+ CD11c− cells in spleen; increase inCD62hi, CD44int subsets of CD4 and CD8 cells; increase in peritonealCD117 cells; increase TcRbeta/CD38 cells in Peyer's patches; increasedpercentage of TcRbeta+ cells in thymus; increased percentages of CD11b+CD11c+ in lymph node; decreased percentage of B220+ Hi CD23+ cells inperitoneal lavage; decreased percentage of B220+ Med CD23−cells inperitoneal lavage; decreased percentages of CD62L Hi CD44 Dim CD4+ andCD8+ cells in spleen; decreased percentage of B220−CD11b Hi cells;decreased mean percentages of CD4 and CD8 cells in lymph node andspleen; increased memory T cells [increased CD62L lo CD44hi]; decreasedT cell:B cell ratio; decreased naive T cells; decreased CD117 cells inperitoneal lavage; decreased mean percentage of CD8 cells, increasedIgG1 response to ovalbumin challenge; increased IgG2a response toovalbumin challenge; increased mean serum IL-6 response to LPSchallenge; increased TNF alpha response to LPS challenge; increasedserum MCP-1 response to LPS challenge; increased mean serum IgM level;increased serum IgA; increase mean serum IgG1; increased mean serumIgG3; decreased serum IgG1 response to ovalbumin challenge; decreasedserum IgG2a response to ovalbumin challenge; decreased mean serum IgAlevel; decreased serum IgG2a level; decrease in serum IgG3 level;increased skin fibroblast proliferation rate; decreased skin fibroblastproliferation rate; increased mean percent of total body fat and totalfat mass; increased mean body weight; increased mean body length;increased total tissue mass (TTM); increased mean femoral midshaftcortical thickness and cross-sectional area; increased mean vertebraltrabecular bone volume, number and connectivity density; decreased meanpercent of total body fat and total fat mass; decreased mean bodyweight; decreased mean body length; decreased total tissue mass (TTM);decreased lean body mass (LBM); decreased femoral bone mineral density(BMD); decreased vertebral bone mineral density (BMD); decreased bonemineral density (BMD) in total body, femur and vertebrae; decreased bonemineral content (BMC); decreased bone mineral density index; decreasedvolumetric bone mineral density (vBMD); decreased mean femoral midshaftcortical thickness and cross-sectional area; decreased mean vertebraltrabecular bone volume, number and connectivity density; osteopetrosis;osteoporosis; chronic inflammation in various tissues; bilateralhydronephrosis (moderate to severe) and inflammation; “pear shapedabdomen”; bilaterally enlarged kidneys, suggesting polycystic kidneydisease; degeneration of the Organ of Corti; hepatocellular dysfunction;biliary obstruction; hepatosplenomegaly characterized by histiocyticinfiltrate; histiocytosis in the small intestine, lymph nodes andspleen; splenomegaly, lymphadenopathy and lymphadenopathy; hyperplasiaof adenoid and tonsils; mild-moderate extra medullary hematopoiesis;homozygous mice were small, dehydrated and exhibited decreasedsubcutaneous fat depots; lipopenia; ulcerous colitis; diffuse markeddegeneration of sensory cochlear hair cells in the inner ear,characterized by a complete loss of both inner and outer cochlear haircells on the basilar membrane; gastric mucosal hyperplasia and chronicinflammation; in creased stomach weight; defective spermatogenesis inthe testes; hypospermia and defective spermatozoa in the epididymus;male infertility; lysosomal storage disease; anemia; growth retardation;reduced viability; perinatal lethality with decreased lymphocytes andlipopenia; homozygous embryonic lethality; and heterozygous embryoniclethality.

The invention also provides an agent which modulates the phenotypeassociated with gene disruption. In one aspect, the agent is an agonistor antagonist of a PRO226, PRO257, PRO268, PRO290, PRO36006, PRO363,PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155,PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474,PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348,PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800,PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196,PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161polypeptide. In yet another aspect, the agonist agent is an anti-PRO226,anti-PRO257, anti-PRO268, anti-PRO290, anti-PRO36006, anti-PRO363,anti-PRO365, anti-PRO382, anti-PRO444, anti-PRO705, anti-PRO1071,anti-PRO1125, anti-PRO1134, anti-PRO1155, anti-PRO1281, anti-PRO1343,anti-PRO1379, anti-PRO1380, anti-PRO1387, anti-PRO1419, anti-PRO1433,anti-PRO1474, anti-PRO1550, anti-PRO1571, anti-PRO1572, anti-PRO1759,anti-PRO1904, anti-PRO35193, anti-PRO4341, anti-PRO4348, anti-PRO4369,anti-PRO4381, anti-PRO4407, anti-PRO4425, anti-PRO4985, anti-PRO4989,anti-PRO5737, anti-PRO5800, anti-PRO5993, anti-PRO6017, anti-PRO7174,anti-PRO9744, anti-PRO9821, anti-PRO9852, anti-PRO9873, anti-PRO10196,anti-PRO34778, anti-PRO20233, anti-PRO21956, anti-PRO57290,anti-PRO38465, anti-PRO38683 or anti-PRO85161 antibody. In still anotheraspect, the antagonist agent is an anti-PRO226, anti-PRO257,anti-PRO268, anti-PRO290, anti-PRO36006, anti-PRO363, anti-PRO365,anti-PRO382, anti-PRO444, anti-PRO705, anti-PRO1071, anti-PRO1125,anti-PRO1134, anti-PRO1155, anti-PRO1281, anti-PRO1343, anti-PRO1379,anti-PRO1380, anti-PRO1387, anti-PRO1419, anti-PRO1433, anti-PRO1474,anti-PRO1550, anti-PRO1571, anti-PRO1572, anti-PRO1759, anti-PRO1904,anti-PRO35193, anti-PRO4341, anti-PRO4348, anti-PRO4369, anti-PRO4381,anti-PRO4407, anti-PRO4425, anti-PRO4985, anti-PRO4989, anti-PRO5737,anti-PRO5800, anti-PRO5993, anti-PRO6017, anti-PRO7174, anti-PRO9744,anti-PRO9821, anti-PRO9852, anti-PRO9873, anti-PRO10196, anti-PRO34778,anti-PRO20233, anti-PRO21956, anti-PRO57290, anti-PRO38465,anti-PRO38683 or anti-PRO85161 antibody.

The invention also provides a method of identifying an agent thatmodulates a physiological characteristic associated with a disruption ofthe gene which encodes for a PRO226, PRO257, PRO268, PRO290, PRO36006,PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125, PRO1134,PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433,PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341,PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737,PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873,PRO10196, PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 orPRO85161 polypeptide, the method comprising:

(a) providing a non-human transgenic animal whose genome comprises adisruption of the gene which encodes for a PRO226, PRO257, PRO268,PRO290, PRO36006, PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071,PRO1125, PRO1134, PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387,PRO1419, PRO1433, PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904,PRO35193, PRO4341, PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985,PRO4989, PRO5737, PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821,PRO9852, PRO9873, PRO10196, PRO34778, PRO20233, PRO21956, PRO57290,PRO38465, PRO38683 or PRO85161 polypeptide;

(b) measuring a physiological characteristic exhibited by the non-humantransgenic animal of (a);

(c) comparing the measured physiological characteristic of (b) with thatof a gender matched wild-type animal, wherein the physiologicalcharacteristic exhibited by the non-human transgenic animal that differsfrom the physiological characteristic exhibited by the wild-type animalis identified as a physiological characteristic associated with genedisruption;

(d) administering a test agent to the non-human transgenic animal of(a); and

(e) determining whether the physiological characteristic associated withgene disruption is modulated.

In one aspect, the non-human transgenic animal exhibits at least one ofthe following physiological characteristics compared with gender matchedwild-type littermates:

In another aspect, the non-human transgenic animal exhibits at least oneof the following physiological characteristics compared with gendermatched wild-type littermates: increased anxiety-like response duringopen field testing; decreased anxiety during open field testing;decreased locomotor activity during open field testing; abnormalcircadian rhythm during home-cage activity testing (low activity duringthe light phase); abnormal circadian rhythm during home-cage activitytesting including decreased ambulatory counts; hypoactivity with nocircadian rhythm; abnormal circadian rhythm during home-cage activitytesting including increased ambulatory counts; increased stress inducedhyperthermia; decreased stress induced hyperthermia; impaired motorcoordination during inverted screen testing; increased immobility intail suspension (increased depressive-like response); increaseddepressive-like response during tail suspension testing; increasedimmobility or decreased depressive-like response during tail suspensiontesting; decreased startle response during prepulse inhibition testing;no startle response indicating deafness; or impaired hearing; decreasedprepulse inhibition with impaired sensorimotor gating/attention;decreased responsiveness in hot plate testing; decreased latency torespond in hot plate testing; opthamological abnormalities; increasedmean artery-to-vein ratio; resistance to pupil dilating drugcyclopentolate hydrochloride; squinty eyes; squint eyes with whitespots; cataracts; retinal degeneration; impaired vision; decreased basalbody temperature; decreased heart rate; increased mean systolic bloodpressure; increased insulin sensitivity; increased mean fasting serumglucose levels; decreased mean serum glucose levels; increased meanserum cholesterol levels; decreased mean serum cholesterol levels;increased mean serum triglyceride levels; decreased mean serumtriglyceride levels; enhanced glucose tolerance; impaired glucosetolerance; decreased mean serum insulin levels; increased mean serumcalcium; increased urobilinogen, notable lipemia; increased albumin,alanine amino transferase, phosphorus and potassium levels; increasedmean serum alkaline phosphatase levels; increased blood urea nitrogen;increased percentage of granulocyte; increased total white blood cell(WBC) count; increased mean absolute neutrophil count; neutropenia;increased absolute lymphocyte count; increased absolute monocyte count;increased monocytes and DC in spleen (CD11b+, CD11b+c+); increased meanplatelet count; increased natural killer (NK) cells in lymph node;decreased neutrophil count; decreased natural killer (NK) cells;decreased mean red blood cell (RBC) count, hemoglobin concentration, andhematocrit; increased mean red cell distribution width; decreased meancorpuscular volume and mean corpuscular hemoglobin; decreased meanplatelet count and increased platelet volume; increase B cell number inlymph node; increase in B cell subtypes in Peyer's patches; increasedpercentage of B cells in lymph node; increase CD25+ cells; increasedthymic DN, decreased DP T cells; increased CD19+ cells in lymph node;increased CD117 in bone marrow cells; increased mean percentage of CD4cells; increased CD8 cells and decrease in B cells; increased percentageCD11b+ cells in peritoneal lavage; increased percentage of B220+ CD11bLow CD23− cells; increased percentages of B220− CD11 Low and CD11b−cells in peritoneal lavage; increased percentage of B220−CD11bhi cellsin peritoneal lavage; decreased percentage of B220+ CD11b− CD23+ cellsin peritoneal lavage; increased percentage of B220− CD43 Hi cells inbone marrow; increased CD11b+ CD11c− cells in spleen; increase inCD62hi, CD44int subsets of CD4 and CD8 cells; increase in peritonealCD117 cells; increase TcRbeta/CD38 cells in Peyer's patches; increasedpercentage of TcRbeta+ cells in thymus; increased percentages of CD11b+CD11c+ in lymph node; decreased percentage of B220+ Hi CD23+ cells inperitoneal lavage; decreased percentage of B220+ Med CD23−cells inperitoneal lavage; decreased percentages of CD62L Hi CD44 Dim CD4+ andCD8+ cells in spleen; decreased percentage of B220−CD11b Hi cells;decreased mean percentages of CD4 and CD8 cells in lymph node andspleen; increased memory T cells [increased CD62L lo CD44hi]; decreasedT cell:B cell ratio; decreased naive T cells; decreased CD117 cells inperitoneal lavage; decreased mean percentage of CD8 cells, increasedIgG1 response to ovalbumin challenge; increased IgG2a response toovalbumin challenge; increased mean serum IL-6 response to LPSchallenge; increased TNF alpha response to LPS challenge; increasedserum MCP-1 response to LPS challenge; increased mean serum IgM level;increased serum IgA; increase mean serum IgG1; increased mean serumIgG3; decreased serum IgG1 response to ovalbumin challenge; decreasedserum IgG2a response to ovalbumin challenge; decreased mean serum IgAlevel; decreased serum IgG2a level; decrease in serum IgG3 level;increased skin fibroblast proliferation rate; decreased skin fibroblastproliferation rate; increased mean percent of total body fat and totalfat mass; increased mean body weight; increased mean body length;increased total tissue mass (TTM); increased mean femoral midshaftcortical thickness and cross-sectional area; increased mean vertebraltrabecular bone volume, number and connectivity density; decreased meanpercent of total body fat and total fat mass; decreased mean bodyweight; decreased mean body length; decreased total tissue mass (TTM);decreased lean body mass (LBM); decreased femoral bone mineral density(BMD); decreased vertebral bone mineral density (BMD); decreased bonemineral density (BMD) in total body, femur and vertebrae; decreased bonemineral content (BMC); decreased bone mineral density index; decreasedvolumetric bone mineral density (vBMD); decreased mean femoral midshaftcortical thickness and cross-sectional area; decreased mean vertebraltrabecular bone volume, number and connectivity density; osteopetrosis;osteoporosis; chronic inflammation in various tissues; bilateralhydronephrosis (moderate to severe) and inflammation; “pear shapedabdomen”; bilaterally enlarged kidneys, suggesting polycystic kidneydisease; degeneration of the Organ of Corti; hepatocellular dysfunction;biliary obstruction; hepatosplenomegaly characterized by histiocyticinfiltrate; histiocytosis in the small intestine, lymph nodes andspleen; splenomegaly, lymphadenopathy and lymphadenopathy; hyperplasiaof adenoid and tonsils; mild-moderate extra medullary hematopoiesis;homozygous mice were small, dehydrated and exhibited decreasedsubcutaneous fat depots; lipopenia; ulcerous colitis; diffuse markeddegeneration of sensory cochlear hair cells in the inner ear,characterized by a complete loss of both inner and outer cochlear haircells on the basilar membrane; gastric mucosal hyperplasia and chronicinflammation; in creased stomach weight; defective spermatogensis in thetestes; hypospermia and defective spermatozoa in the epididymus; maleinfertility; lysosomal storage disease; anemia; growth retardation;reduced viability; perinatal lethality with decreased lymphocytes andlipopenia; homozygous embryonic lethality; and heterozygous embryoniclethality.

The invention also provides an agent that modulates a physiologicalcharacteristic which is associated with gene disruption. In one aspect,the agent is an agonist or antagonist of the phenotype associated with adisruption of a gene which encodes for a PRO226, PRO257, PRO268, PRO290,PRO36006, PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125,PRO1134, PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419,PRO1433, PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193,PRO4341, PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989,PRO5737, PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852,PRO9873, PRO10196, PRO34778, PRO20233, PRO21956, PRO57290, PRO38465,PRO38683 or PRO85161 polypeptide. In yet another aspect, the agent is anagonist or antagonist of a PRO226, PRO257, PRO268, PRO290, PRO36006,PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125, PRO1134,PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433,PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341,PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737,PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873,PRO10196, PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 orPRO85161 polypeptide. In yet another aspect, the agonist agent is ananti-PRO226, anti-PRO257, anti-PRO268, anti-PRO290, anti-PRO36006,anti-PRO363, anti-PRO365, anti-PRO382, anti-PRO444, anti-PRO705,anti-PRO1071, anti-PRO1125, anti-PRO1134, anti-PRO1155, anti-PRO1281,anti-PRO1343, anti-PRO1379, anti-PRO1380, anti-PRO1387, anti-PRO1419,anti-PRO1433, anti-PRO1474, anti-PRO1550, anti-PRO1571, anti-PRO1572,anti-PRO1759, anti-PRO1904, anti-PRO35193, anti-PRO4341, anti-PRO4348,anti-PRO4369, anti-PRO4381, anti-PRO4407, anti-PRO4425, anti-PRO4985,anti-PRO4989, anti-PRO5737, anti-PRO5800, anti-PRO5993, anti-PRO6017,anti-PRO7174, anti-PRO9744, anti-PRO9821, anti-PRO9852, anti-PRO9873,anti-PRO10196, anti-PRO34778, anti-PRO20233, anti-PRO21956,anti-PRO57290, anti-PRO38465, anti-PRO38683 or anti-PRO85161 antibody.In still another aspect, the antagonist agent is an anti-PRO226,anti-PRO257, anti-PRO268, anti-PRO290, anti-PRO36006, anti-PRO363,anti-PRO365, anti-PRO382, anti-PRO444, anti-PRO705, anti-PRO1071,anti-PRO1125, anti-PRO1134, anti-PRO1155, anti-PRO1281, anti-PRO1343,anti-PRO1379, anti-PRO1380, anti-PRO1387, anti-PRO1419, anti-PRO1433,anti-PRO1474, anti-PRO1550, anti-PRO1571, anti-PRO1572, anti-PRO1759,anti-PRO1904, anti-PRO35193, anti-PRO4341, anti-PRO4348, anti-PRO4369,anti-PRO4381, anti-PRO4407, anti-PRO4425, anti-PRO4985, anti-PRO4989,anti-PRO5737, anti-PRO5800, anti-PRO5993, anti-PRO6017, anti-PRO7174,anti-PRO9744, anti-PRO9821, anti-PRO9852, anti-PRO9873, anti-PRO10196,anti-PRO34778, anti-PRO20233, anti-PRO21956, anti-PRO57290,anti-PRO38465, anti-PRO38683 or anti-PRO85161 antibody.

The invention also provides a method of identifying an agent whichmodulates a behavior associated with a disruption of the gene whichencodes for a PRO226, PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365,PRO382, PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281,PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550,PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369,PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993,PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196,PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161polypeptide, the method comprising:

(a) providing a non-human transgenic animal whose genome comprises adisruption of the gene which encodes for a PRO226, PRO257, PRO268,PRO290, PRO36006, PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071,PRO1125, PRO1134, PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387,PRO1419, PRO1433, PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904,PRO35193, PRO4341, PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985,PRO4989, PRO5737, PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821,PRO9852, PRO9873, PRO10196, PRO34778, PRO20233, PRO21956, PRO57290,PRO38465, PRO38683 or PRO85161 polypeptide;

(b) observing the behavior exhibited by the non-human transgenic animalof (a);

(c) comparing the observed behavior of (b) with that of a gender matchedwild-type animal, wherein the observed behavior exhibited by thenon-human transgenic animal that differs from the observed behaviorexhibited by the wild-type animal is identified as a behavior associatedwith gene disruption;

(d) administering a test agent to the non-human transgenic animal of(a); and

(e) determining whether the agent modulates the behavior associated withgene disruption.

In one aspect, the observed behavior is an increased anxiety-likeresponse during open field activity testing. In yet another aspect, theobserved behavior is a decreased anxiety-like response during open fieldactivity testing. In yet another aspect, the observed behavior is anabnormal circadian rhythm during home-cage activity testing. In yetanother aspect, the observed behavior is an enhanced motor coordinationduring inverted screen testing. In yet another aspect, the observedbehavior is impaired motor coordination during inverted screen testing.In yet another aspect, the observed behavior includes depression,generalized anxiety disorders, attention deficit disorder, sleepdisorder, hyperactivity disorder, obsessive compulsive disorder,schizophrenia, cognitive disorders, hyperalgesia and sensory disorders.Such disorders include the category defined as “anxiety disorders” whichinclude but are not limited to: mild to moderate anxiety, anxietydisorder due to a general medical condition, anxiety disorder nototherwise specified, generalized anxiety disorder, panic attack, panicdisorder with agoraphobia, panic disorder without agoraphobia,posttraumatic stress disorder, social phobia, social anxiety, autism,specific phobia, substance-induced anxiety disorder, acute alcoholwithdrawal, obsessive compulsive disorder, agoraphobia, monopolardisorders, bipolar disorder I or II, bipolar disorder not otherwisespecified, cyclothymic disorder, depressive disorder, major depressivedisorder, mood disorder, substance-induced mood disorder, enhancement ofcognitive function, loss of cognitive function associated with but notlimited to Alzheimer's disease, stroke, or traumatic injury to thebrain, seizures resulting from disease or injury including but notlimited to epilepsy, learning disorders/disabilities, cerebral palsy. Inaddition, anxiety disorders may apply to personality disorders includingbut not limited to the following types: paranoid, antisocial, avoidantbehavior, border line personality disorders, dependent, histrionic,narcissistic, obsessive-compulsive, schizoid, and schizotypal.

The invention also provides an agent that modulates a behavior which isassociated with gene disruption. In one aspect, the agent is an agonistor antagonist of the phenotype associated with a disruption of a genewhich encodes for a PRO226, PRO257, PRO268, PRO290, PRO36006, PRO363,PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155,PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474,PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348,PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800,PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196,PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161polypeptide. In yet another aspect, the agent is an agonist orantagonist of a PRO226, PRO257, PRO268, PRO290, PRO36006, PRO363,PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155,PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474,PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348,PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800,PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196,PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161polypeptide. In yet another aspect, the agonist agent is an anti-PRO226,anti-PRO257, anti-PRO268, anti-PRO290, anti-PRO36006, anti-PRO363,anti-PRO365, anti-PRO382, anti-PRO444, anti-PRO705, anti-PRO1071,anti-PRO1125, anti-PRO1134, anti-PRO1155, anti-PRO1281, anti-PRO1343,anti-PRO1379, anti-PRO1380, anti-PRO1387, anti-PRO1419, anti-PRO1433,anti-PRO1474, anti-PRO1550, anti-PRO1571, anti-PRO1572, anti-PRO1759,anti-PRO1904, anti-PRO35193, anti-PRO4341, anti-PRO4348, anti-PRO4369,anti-PRO4381, anti-PRO4407, anti-PRO4425, anti-PRO4985, anti-PRO4989,anti-PRO5737, anti-PRO5800, anti-PRO5993, anti-PRO6017, anti-PRO7174,anti-PRO9744, anti-PRO9821, anti-PRO9852, anti-PRO9873, anti-PRO10196,anti-PRO34778, anti-PRO20233, anti-PRO21956, anti-PRO57290,anti-PRO38465, anti-PRO38683 or anti-PRO85161 antibody. In still anotheraspect, the antagonist agent is an anti-PRO226, anti-PRO257,anti-PRO268, anti-PRO290, anti-PRO36006, anti-PRO363, anti-PRO365,anti-PRO382, anti-PRO444, anti-PRO705, anti-PRO1071, anti-PRO1125,anti-PRO1134, anti-PRO1155, anti-PRO1281, anti-PRO1343, anti-PRO1379,anti-PRO1380, anti-PRO1387, anti-PRO1419, anti-PRO1433, anti-PRO1474,anti-PRO1550, anti-PRO1571, anti-PRO1572, anti-PRO1759, anti-PRO1904,anti-PRO35193, anti-PRO4341, anti-PRO4348, anti-PRO4369, anti-PRO4381,anti-PRO4407, anti-PRO4425, anti-PRO4985, anti-PRO4989, anti-PRO5737,anti-PRO5800, anti-PRO5993, anti-PRO6017, anti-PRO7174, anti-PRO9744,anti-PRO9821, anti-PRO9852, anti-PRO9873, anti-PRO10196, anti-PRO34778,anti-PRO20233, anti-PRO21956, anti-PRO57290, anti-PRO38465,anti-PRO38683 or anti-PRO85161 antibody.

The invention also provides a method of identifying an agent thatameliorates or modulates a neurological disorder; a cardiovascular,endothelial or angiogenic disorder; an eye abnormality; an immunologicaldisorder; an oncological disorder; a bone metabolic abnormality ordisorder; a lipid metabolic disorder; or a developmental abnormalityassociated with a disruption in the gene which encodes for a PRO226,PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382, PRO444,PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343, PRO1379,PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571, PRO1572,PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381, PRO4407,PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017, PRO7174,PRO9744, PRO9821, PRO9852, PRO9873, PRO10196, PRO34778, PRO20233,PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161 polypeptide, themethod comprising:

(a) providing a non-human transgenic animal whose genome comprises adisruption of the gene which encodes for a PRO226, PRO257, PRO268,PRO290, PRO36006, PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071,PRO1125, PRO1134, PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387,PRO1419, PRO1433, PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904,PRO35193, PRO4341, PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985,PRO4989, PRO5737, PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821,PRO9852, PRO9873, PRO10196, PRO34778, PRO20233, PRO21956, PRO57290,PRO38465, PRO38683 or PRO85161 polypeptide;

(b) administering a test agent to said non-human transgenic animal; and

(c) determining whether the test agent ameliorates or modulates theneurological disorder; cardiovascular, endothelial or angiogenicdisorder; eye abnormality; immunological disorder; oncological disorder;bone metabolic abnormality or disorder; lipid metabolic disorder; ordevelopmental abnormality associated with the gene disruption in thenon-human transgenic animal.

In yet another aspect, the neurological disorder is an increasedanxiety-like response during open field activity testing. In yet anotheraspect, the neurological disorder is a decreased anxiety-like responseduring open field activity testing. In yet another aspect, theneurological disorder is an abnormal circadian rhythm during home-cageactivity testing. In yet another aspect, the neurological disorder is anenhanced motor coordination during inverted screen testing. In yetanother aspect, the neurological disorder is impaired motor coordinationduring inverted screen testing. In yet another aspect, the neurologicaldisorder includes depression, generalized anxiety disorders, attentiondeficit disorder, sleep disorder, hyperactivity disorder, obsessivecompulsive disorder, schizophrenia, cognitive disorders, hyperalgesiaand sensory disorders. Such neurological disorders include the categorydefined as “anxiety disorders” which include but are not limited to:mild to moderate anxiety, anxiety disorder due to a general medicalcondition, anxiety disorder not otherwise specified, generalized anxietydisorder, panic attack, panic disorder with agoraphobia, panic disorderwithout agoraphobia, posttraumatic stress disorder, social phobia,social anxiety, autism, specific phobia, substance-induced anxietydisorder, acute alcohol withdrawal, obsessive compulsive disorder,agoraphobia, monopolar disorders, bipolar disorder I or II, bipolardisorder not otherwise specified, cyclothymic disorder, depressivedisorder, major depressive disorder, mood disorder, substance-inducedmood disorder, enhancement of cognitive function, loss of cognitivefunction associated with but not limited to Alzheimer's disease, stroke,or traumatic injury to the brain, seizures resulting from disease orinjury including but not limited to epilepsy, learningdisorders/disabilities, cerebral palsy. In addition, anxiety disordersmay apply to personality disorders including but not limited to thefollowing types: paranoid, antisocial, avoidant behavior, borderlinepersonality disorders, dependent, histrionic, narcissistic,obsessive-compulsive, schizoid, and schizotypal.

In another aspect, the eye abnormality is a retinal abnormality. Instill another aspect, the eye abnormality is consistent with visionproblems or blindness. In yet another aspect, the retinal abnormality isconsistent with retinitis pigmentosa or is characterized by retinaldegeneration or retinal dysplasia.

In still another aspect, the retinal abnormalities the retinalabnormalities are consistent with retinal dysplasia, variousretinopathies, including retinopathy of prematurity, retrolentalfibroplasia, neovascular glaucoma, age-related macular degeneration,diabetic macular edema, corneal neovascularization, corneal graftneovascularization, corneal graft rejection, retinal/choroidalneovascularization, neovascularization of the angle (rubeosis), ocularneovascular disease, vascular restenosis, arteriovenous malformations(AVM), meningioma, hemangioma, angiofibroma, thyroid hyperplasias(including Grave's disease), corneal and other tissue transplantation,retinal artery obstruction or occlusion; retinal degeneration causingsecondary atrophy of the retinal vasculature, retinitis pigmentosa,macular dystrophies, Stargardt's disease, congenital stationary nightblindness, choroideremia, gyrate atrophy, Leber's congenital amaurosis,retinoschisis disorders, Wagner's syndrome, Usher syndromes, Zellwegersyndrome, Saldino-Mainzer syndrome, Senior-Loken syndrome, Bardet-Biedlsyndrome, Alport's syndrome, Alstrom's syndrome, Cockayne's syndrome,dysplaisa spondyloepiphysaria congentia, Flynn-Aird syndrome, Friedreichataxia, Hallgren syndrome, Marshall syndrome, Albers-Schnoberg disease,Refsum's disease, Kearns-Sayre syndrome, Waardenburg's syndrome, Alagilesyndrome, myotonic dystrophy, olivopontocerebellar atrophy, Pierre-Mariedunsdrome, Stickler syndrome, carotinemeia, cystinosis, Wolframsyndrome, Bassen-Kornzweig syndrome, abetalipoproteinemia, incontinentiapigmenti, Batten's disease, mucopolysaccharidoses, homocystinuria, ormannosidosis.

In still another aspect, the eye abnormality is a cataract. In still yetanother aspect, the cataract is a systemic disease such as human Down'ssyndrome, Hallerman-Streiff syndrome, Lowe syndrome, galactosemia,Marfan syndrome, Trismoy 13-15, Alport syndrome, myotonic dystrophy,Fabry disease, hypoparathroidism, or Conradi syndrome.

In still another aspect, the developmental abnormality comprisesembryonic lethality or reduced viability.

In yet another aspect, the cardiovascular, endothelial or angiogenicdisorders are arterial diseases, such as diabetes mellitus; papilledema;optic atrophy; atherosclerosis; angina; myocardial infarctions such asacute myocardial infarctions, cardiac hypertrophy, and heart failuresuch as congestive heart failure; hypertension; inflammatoryvasculitides; Reynaud's disease and Reynaud's phenomenon; aneurysms andarterial restenosis; venous and lymphatic disorders such asthrombophlebitis, lymphangitis, and lymphedema; peripheral vasculardisease; cancer such as vascular tumors, e.g., hemangioma (capillary andcavernous), glomus tumors, telangiectasia, bacillary angiomatosis,hemangioendothelioma, angiosarcoma, haemangiopericytoma, Kaposi'ssarcoma, lymphangioma, and lymphangiosarcoma; tumor angiogenesis; traumasuch as wounds, burns, and other injured tissue, implant fixation,scarring; ischemia reperfusion injury; rheumatoid arthritis;cerebrovascular disease; renal diseases such as acute renal failure, orosteoporosis.

In still yet another aspect, the immunological disorders are consistentwith systemic lupus erythematosis; rheumatoid arthritis; juvenilechronic arthritis; spondyloarthropathies; systemic sclerosis(scleroderma); idiopathic inflammatory myopathies (dermatomyositis,polymyositis); Sjögren's syndrome; systemic vasculitis; sarcoidosis;autoimmune hemolytic anemia (immune pancytopenia, paroxysmal nocturnalhemoglobinuria); autoimmune thrombocytopenia (idiopathicthrombocytopenic purpura, immune-mediated thrombocytopenia); thyroiditis(Grave's disease, Hashimoto's thyroiditis, juvenile lymphocyticthyroiditis, atrophic thyroiditis); diabetes mellitus; immune-mediatedrenal disease (glomerulonephritis, tubulointerstitial nephritis);demyelinating diseases of the central and peripheral nervous systemssuch as multiple sclerosis, idiopathic demyelinating polyneuropathy orGuillain-Barré syndrome, and chronic inflammatory demyelinatingpolyneuropathy; hepatobiliary diseases such as infectious hepatitis(hepatitis A, B, C, D, E and other non-hepatotropic viruses), autoimmunechronic active hepatitis, primary biliary cirrhosis, granulomatoushepatitis, and sclerosing cholangitis; inflammatory bowel disease(ulcerative colitis: Crohn's disease); gluten-sensitive enteropathy, andWhipple's disease; autoimmune or immune-mediated skin diseases includingbullous skin diseases, erythema multiforme and contact dermatitis,psoriasis; allergic diseases such as asthma, allergic rhinitis, atopicdermatitis, food hypersensitivity and urticaria; immunologic diseases ofthe lung such as eosinophilic pneumonia, idiopathic pulmonary fibrosisand hypersensitivity pneumonitis; or transplantation associated diseasesincluding graft rejection and graft-versus-host disease.

In yet another aspect, the bone metabolic abnormality or disorder isarthritis, osteoporosis, osteopenia or osteopetrosis.

In another aspect, the non-human transgenic animal exhibits at least oneof the following physiological characteristics compared with gendermatched wild-type littermates: increased anxiety-like response duringopen field testing; decreased anxiety during open field testing;decreased locomotor activity during open field testing; abnormalcircadian rhythm during home-cage activity testing (low activity duringthe light phase); abnormal circadian rhythm during home-cage activitytesting including decreased ambulatory counts; hypoactivity with nocircadian rhythm; abnormal circadian rhythm during home-cage activitytesting including increased ambulatory counts; increased stress inducedhyperthermia; decreased stress induced hyperthermia; impaired motorcoordination during inverted screen testing; increased immobility intail suspension (increased depressive-like response); increaseddepressive-like response during tail suspension testing; increasedimmobility or decreased depressive-like response during tail suspensiontesting; decreased startle response during prepulse inhibition testing;no startle response indicating deafness; or impaired hearing; decreasedprepulse inhibition with impaired sensorimotor gating/attention;decreased responsiveness in hot plate testing; decreased latency torespond in hot plate testing; opthamological abnormalities; increasedmean artery-to-vein ratio; resistance to pupil dilating drugcyclopentolate hydrochloride; squinty eyes; squint eyes with whitespots; cataracts; retinal degeneration; impaired vision; decreased basalbody temperature; decreased heart rate; increased mean systolic bloodpressure; increased insulin sensitivity; increased mean fasting serumglucose levels; decreased mean serum glucose levels; increased meanserum cholesterol levels; decreased mean serum cholesterol levels;increased mean serum triglyceride levels; decreased mean serumtriglyceride levels; enhanced glucose tolerance; impaired glucosetolerance; decreased mean serum insulin levels; increased mean serumcalcium; increased urobilinogen, notable lipemia; increased albumin,alanine amino transferase, phosphorus and potassium levels; increasedmean serum alkaline phosphatase levels; increased blood urea nitrogen;increased percentage of granulocyte; increased total white blood cell(WBC) count; increased mean absolute neutrophil count; neutropenia;increased absolute lymphocyte count; increased absolute monocyte count;increased monocytes and DC in spleen (CD11b+, CD11b+c+); increased meanplatelet count; increased natural killer (NK) cells in lymph node;decreased neutrophil count; decreased natural killer (NK) cells;decreased mean red blood cell (RBC) count, hemoglobin concentration, andhematocrit; increased mean red cell distribution width; decreased meancorpuscular volume and mean corpuscular hemoglobin; decreased meanplatelet count and increased platelet volume; increase B cell number inlymph node; increase in B cell subtypes in Peyer's patches; increasedpercentage of B cells in lymph node; increase CD25+ cells; increasedthymic DN, decreased DP T cells; increased CD19+ cells in lymph node;increased CD117 in bone marrow cells; increased mean percentage of CD4cells; increased CD8 cells and decrease in B cells; increased percentageCD11b+ cells in peritoneal lavage; increased percentage of B220+CD11bLow CD23− cells; increased percentages of B220− CD11 Low and CD11b−cells in peritoneal lavage; increased percentage of B220−CD11bHi cellsin peritoneal lavage; decreased percentage of B220+ CD11b− CD23+ cellsin peritoneal lavage; increased percentage of B220− CD43 Hi cells inbone marrow; increased CD11b+ CD11c− cells in spleen; increase inCD62hi, CD44int subsets of CD4 and CD8 cells; increase in peritonealCD117 cells; increase TcRbeta/CD38 cells in Peyer's patches; increasedpercentage of TcRbeta+ cells in thymus; increased percentages of CD11b+CD11c+ in lymph node; decreased percentage of B220+ Hi CD23+ cells inperitoneal lavage; decreased percentage of B220+ Med CD23−cells inperitoneal lavage; decreased percentages of CD62L Hi CD44 Dim CD4+ andCD8+ cells in spleen; decreased percentage of B220−CD11b Hi cells;decreased mean percentages of CD4 and CD8 cells in lymph node andspleen; increased memory T cells [increased CD62L lo CD44hi]; decreasedT cell:B cell ratio; decreased naive T cells; decreased CD117 cells inperitoneal lavage; decreased mean percentage of CD8 cells, increasedIgG1 response to ovalbumin challenge; increased IgG2a response toovalbumin challenge; increased mean serum IL-6 response to LPSchallenge; increased TNF alpha response to LPS challenge; increasedserum MCP-1 response to LPS challenge; increased mean serum IgM level;increased serum IgA; increase mean serum IgG1; increased mean serumIgG3; decreased serum IgG1 response to ovalbumin challenge; decreasedserum IgG2a response to ovalbumin challenge; decreased mean serum IgAlevel; decreased serum IgG2a level; decrease in serum IgG3 level;increased skin fibroblast proliferation rate; decreased skin fibroblastproliferation rate; increased mean percent of total body fat and totalfat mass; increased mean body weight; increased mean body length;increased total tissue mass (TTM); increased mean femoral midshaftcortical thickness and cross-sectional area; increased mean vertebraltrabecular bone volume, number and connectivity density; decreased meanpercent of total body fat and total fat mass; decreased mean bodyweight; decreased mean body length; decreased total tissue mass (TTM);decreased lean body mass (LBM); decreased femoral bone mineral density(BMD); decreased vertebral bone mineral density (BMD); decreased bonemineral density (BMD) in total body, femur and vertebrae; decreased bonemineral content (BMC); decreased bone mineral density index; decreasedvolumetric bone mineral density (vBMD); decreased mean femoral midshaftcortical thickness and cross-sectional area; decreased mean vertebraltrabecular bone volume, number and connectivity density; osteopetrosis;osteoporosis; chronic inflammation in various tissues; bilateralhydronephrosis (moderate to severe) and inflammation; “pear shapedabdomen”; bilaterally enlarged kidneys, suggesting polycystic kidneydisease; degeneration of the Organ of Corti; hepatocellular dysfunction;biliary obstruction; hepatosplenomegaly characterized by histiocyticinfiltrate; histiocytosis in the small intestine, lymph nodes andspleen; splenomegaly, lymphadenopathy and lymphadenopathy; hyperplasiaof adenoid and tonsils; mild-moderate extra medullary hematopoiesis;homozygous mice were small, dehydrated and exhibited decreasedsubcutaneous fat depots; lipopenia; ulcerous colitis; diffuse markeddegeneration of sensory cochlear hair cells in the inner ear,characterized by a complete loss of both inner and outer cochlear haircells on the basilar membrane; gastric mucosal hyperplasia and chronicinflammation; in creased stomach weight; defective spermatogensis in thetestes; hypospermia and defective spermatozoa in the epididymus; maleinfertility; lysosomal storage disease; anemia; growth retardation;reduced viability; perinatal lethality with decreased lymphocytes andlipopenia; homozygous embryonic lethality; and heterozygous embryoniclethality.

The invention also provides an agent that ameliorates or modulates aneurological disorder; a cardiovascular, endothelial or angiogenicdisorder; an eye abnormality; an immunological disorder; an oncologicaldisorder; a bone metabolic abnormality or disorder; a lipid metabolicdisorder; or a developmental abnormality which is associated with genedisruption. In one aspect, the agent is an agonist or antagonist of thephenotype associated with a disruption of a gene which encodes for aPRO226, PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382,PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343,PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571,PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381,PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017,PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196, PRO34778,PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161polypeptide. In yet another aspect, the agent is an agonist orantagonist of a PRO226, PRO257, PRO268, PRO290, PRO36006, PRO363,PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155,PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474,PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348,PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800,PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196,PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161polypeptide. In yet another aspect, the agonist agent is an anti-PRO226,anti-PRO257, anti-PRO268, anti-PRO290, anti-PRO36006, anti-PRO363,anti-PRO365, anti-PRO382, anti-PRO444, anti-PRO705, anti-PRO1071,anti-PRO1125, anti-PRO1134, anti-PRO1155, anti-PRO1281, anti-PRO1343,anti-PRO1379, anti-PRO1380, anti-PRO1387, anti-PRO1419, anti-PRO1433,anti-PRO1474, anti-PRO1550, anti-PRO1571, anti-PRO1572, anti-PRO1759,anti-PRO1904, anti-PRO35193, anti-PRO4341, anti-PRO4348, anti-PRO4369,anti-PRO4381, anti-PRO4407, anti-PRO4425, anti-PRO4985, anti-PRO4989,anti-PRO5737, anti-PRO5800, anti-PRO5993, anti-PRO6017, anti-PRO7174,anti-PRO9744, anti-PRO9821, anti-PRO9852, anti-PRO9873, anti-PRO10196,anti-PRO34778, anti-PRO20233, anti-PRO21956, anti-PRO57290,anti-PRO38465, anti-PRO38683 or anti-PRO85161 antibody. In still anotheraspect, the antagonist agent is an anti-PRO226, anti-PRO257,anti-PRO268, anti-PRO290, anti-PRO36006, anti-PRO363, anti-PRO365,anti-PRO382, anti-PRO444, anti-PRO705, anti-PRO1071, anti-PRO1125,anti-PRO1134, anti-PRO1155, anti-PRO1281, anti-PRO1343, anti-PRO1379,anti-PRO1380, anti-PRO1387, anti-PRO1419, anti-PRO1433, anti-PRO1474,anti-PRO1550, anti-PRO1571, anti-PRO1572, anti-PRO1759, anti-PRO1904,anti-PRO35193, anti-PRO4341, anti-PRO4348, anti-PRO4369, anti-PRO4381,anti-PRO4407, anti-PRO4425, anti-PRO4985, anti-PRO4989, anti-PRO5737,anti-PRO5800, anti-PRO5993, anti-PRO6017, anti-PRO7174, anti-PRO9744,anti-PRO9821, anti-PRO9852, anti-PRO9873, anti-PRO10196, anti-PRO34778,anti-PRO20233, anti-PRO21956, anti-PRO57290, anti-PRO38465,anti-PRO38683 or anti-PRO85161 antibody.

The invention also provides a therapeutic agent for the treatment of aneurological disorder; a cardiovascular, endothelial or angiogenicdisorder; an eye abnormality; an immunological disorder; an oncologicaldisorder; a bone metabolic abnormality or disorder; a lipid metabolicdisorder; or a developmental abnormality.

The invention also provides a method of identifying an agent thatmodulates the expression of a PRO226, PRO257, PRO268, PRO290, PRO36006,PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125, PRO1134,PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433,PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341,PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737,PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873,PRO10196, PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 orPRO85161 polypeptide, the method comprising:

(a) contacting a test agent with a host cell expressing a PRO226,PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382, PRO444,PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343, PRO1379,PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571, PRO1572,PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381, PRO4407,PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017, PRO7174,PRO9744, PRO9821, PRO9852, PRO9873, PRO10196, PRO34778, PRO20233,PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161 polypeptide; and

(b) determining whether the test agent modulates the expression of thePRO226, PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382,PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343,PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571,PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381,PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017,PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196, PRO34778,PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161 polypeptideby the host cell.

The invention also provides an agent that modulates the expression of aPRO226, PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382,PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343,PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571,PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381,PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017,PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196, PRO34778,PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161polypeptide. In one aspect, the agent is an agonist or antagonist of thephenotype associated with a disruption of a gene which encodes for aPRO226, PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382,PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343,PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571,PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381,PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017,PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196, PRO34778,PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161polypeptide. In yet another aspect, the agent is an agonist orantagonist of a PRO226, PRO257, PRO268, PRO290, PRO36006, PRO363,PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155,PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474,PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348,PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800,PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196,PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161polypeptide. In yet another aspect, the agonist agent is an anti-PRO226,anti-PRO257, anti-PRO268, anti-PRO290, anti-PRO36006, anti-PRO363,anti-PRO365, anti-PRO382, anti-PRO444, anti-PRO705, anti-PRO1071,anti-PRO1125, anti-PRO1134, anti-PRO1155, anti-PRO1281, anti-PRO1343,anti-PRO1379, anti-PRO1380, anti-PRO1387, anti-PRO1419, anti-PRO1433,anti-PRO1474, anti-PRO1550, anti-PRO1571, anti-PRO1572, anti-PRO1759,anti-PRO1904, anti-PRO35193, anti-PRO4341, anti-PRO4348, anti-PRO4369,anti-PRO4381, anti-PRO4407, anti-PRO4425, anti-PRO4985, anti-PRO4989,anti-PRO5737, anti-PRO5800, anti-PRO5993, anti-PRO6017, anti-PRO7174,anti-PRO9744, anti-PRO9821, anti-PRO9852, anti-PRO9873, anti-PRO10196,anti-PRO34778, anti-PRO20233, anti-PRO21956, anti-PRO57290,anti-PRO38465, anti-PRO38683 or anti-PRO85161 antibody. In still anotheraspect, the antagonist agent is an anti-PRO226, anti-PRO257,anti-PRO268, anti-PRO290, anti-PRO36006, anti-PRO363, anti-PRO365,anti-PRO382, anti-PRO444, anti-PRO705, anti-PRO1071, anti-PRO1125,anti-PRO1134, anti-PRO1155, anti-PRO1281, anti-PRO1343, anti-PRO1379,anti-PRO1380, anti-PRO1387, anti-PRO1419, anti-PRO1433, anti-PRO1474,anti-PRO1550, anti-PRO1571, anti-PRO1572, anti-PRO1759, anti-PRO1904,anti-PRO35193, anti-PRO4341, anti-PRO4348, anti-PRO4369, anti-PRO4381,anti-PRO4407, anti-PRO4425, anti-PRO4985, anti-PRO4989, anti-PRO5737,anti-PRO5800, anti-PRO5993, anti-PRO6017, anti-PRO7174, anti-PRO9744,anti-PRO9821, anti-PRO9852, anti-PRO9873, anti-PRO10196, anti-PRO34778,anti-PRO20233, anti-PRO21956, anti-PRO57290, anti-PRO38465,anti-PRO38683 or anti-PRO85161 antibody.

The invention also provides a method of evaluating a therapeutic agentcapable of affecting a condition associated with a disruption of a genewhich encodes for a PRO226, PRO257, PRO268, PRO290, PRO36006, PRO363,PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155,PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474,PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348,PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800,PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196,PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161polypeptide, the method comprising:

(a) providing a non-human transgenic animal whose genome comprises adisruption of the gene which encodes for the PRO226, PRO257, PRO268,PRO290, PRO36006, PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071,PRO1125, PRO1134, PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387,PRO1419, PRO1433, PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904,PRO35193, PRO4341, PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985,PRO4989, PRO5737, PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821,PRO9852, PRO9873, PRO10196, PRO34778, PRO20233, PRO21956, PRO57290,PRO38465, PRO38683 or PRO85161 polypeptide;

(b) measuring a physiological characteristic of the non-human transgenicanimal of (a);

(c) comparing the measured physiological characteristic of (b) with thatof a gender matched wild-type animal, wherein the physiologicalcharacteristic of the non-human transgenic animal that differs from thephysiological characteristic of the wild-type animal is identified as acondition resulting from the gene disruption in the non-human transgenicanimal;

(d) administering a test agent to the non-human transgenic animal of(a); and

(e) evaluating the effects of the test agent on the identified conditionassociated with gene disruption in the non-human transgenic animal.

In one aspect, the condition is a neurological disorder; acardiovascular, endothelial or angiogenic disorder; an eye abnormality;an immunological disorder; an oncological disorder; a bone metabolicabnormality or disorder; a lipid metabolic disorder; or a developmentalabnormality.

The invention also provides a therapeutic agent which is capable ofaffecting a condition associated with gene disruption. In one aspect,the agent is an agonist or antagonist of the phenotype associated with adisruption of a gene which encodes for a PRO226, PRO257, PRO268, PRO290,PRO36006, PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125,PRO1134, PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419,PRO1433, PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193,PRO4341, PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989,PRO5737, PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852,PRO9873, PRO10196, PRO34778, PRO20233, PRO21956, PRO57290, PRO38465,PRO38683 or PRO85161 polypeptide. In yet another aspect, the agent is anagonist or antagonist of a PRO226, PRO257, PRO268, PRO290, PRO36006,PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125, PRO1134,PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433,PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341,PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737,PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873,PRO10196, PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 orPRO85161 polypeptide. In yet another aspect, the agonist agent is ananti-PRO226, anti-PRO257, anti-PRO268, anti-PRO290, anti-PRO36006,anti-PRO363, anti-PRO365, anti-PRO382, anti-PRO444, anti-PRO705,anti-PRO1071, anti-PRO1125, anti-PRO1134, anti-PRO1155, anti-PRO1281,anti-PRO1343, anti-PRO1379, anti-PRO1380, anti-PRO1387, anti-PRO1419,anti-PRO1433, anti-PRO1474, anti-PRO1550, anti-PRO1571, anti-PRO1572,anti-PRO1759, anti-PRO1904, anti-PRO35193, anti-PRO4341, anti-PRO4348,anti-PRO4369, anti-PRO4381, anti-PRO4407, anti-PRO4425, anti-PRO4985,anti-PRO4989, anti-PRO5737, anti-PRO5800, anti-PRO5993, anti-PRO6017,anti-PRO7174, anti-PRO9744, anti-PRO9821, anti-PRO9852, anti-PRO9873,anti-PRO10196, anti-PRO34778, anti-PRO20233, anti-PRO21956,anti-PRO57290, anti-PRO38465, anti-PRO38683 or anti-PRO8516 antibody. Instill another aspect, the antagonist agent is an anti-PRO226,anti-PRO257, anti-PRO268, anti-PRO290, anti-PRO36006, anti-PRO363,anti-PRO365, anti-PRO382, anti-PRO444, anti-PRO705, anti-PRO1071,anti-PRO1125, anti-PRO1134, anti-PRO1155, anti-PRO1281, anti-PRO1343,anti-PRO1379, anti-PRO1380, anti-PRO1387, anti-PRO1419, anti-PRO1433,anti-PRO1474, anti-PRO1550, anti-PRO1571, anti-PRO1572, anti-PRO1759,anti-PRO1904, anti-PRO35193, anti-PRO4341, anti-PRO4348, anti-PRO4369,anti-PRO4381, anti-PRO4407, anti-PRO4425, anti-PRO4985, anti-PRO4989,anti-PRO5737, anti-PRO5800, anti-PRO5993, anti-PRO6017, anti-PRO7174,anti-PRO9744, anti-PRO9821, anti-PRO9852, anti-PRO9873, anti-PRO10196,anti-PRO34778, anti-PRO20233, anti-PRO21956, anti-PRO57290,anti-PRO38465, anti-PRO38683 or anti-PRO8516 antibody.

The invention also provides a pharmaceutical composition comprising atherapeutic agent capable of affecting the condition associated withgene disruption.

The invention also provides a method of treating or preventing orameliorating a neurological disorder; cardiovascular, endothelial orangiogenic disorder; immunological disorder; oncological disorder; bonemetabolic abnormality or disorder, or embryonic lethality associatedwith the disruption of a gene which encodes for a PRO226, PRO257,PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382, PRO444, PRO705,PRO171, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343, PRO1379, PRO1380,PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571, PRO1572, PRO1759,PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381, PRO4407, PRO4425,PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017, PRO7174, PRO9744,PRO9821, PRO9852, PRO9873, PRO10196, PRO34778, PRO20233, PRO21956,PRO57290, PRO38465, PRO38683 or PRO85161 polypeptide, the methodcomprising administering to a subject in need of such treatment whom mayalready have the disorder, or may be prone to have the disorder or maybe in whom the disorder is to be prevented, a therapeutically effectiveamount of a therapeutic agent, or agonists or antagonists thereof,thereby effectively treating or preventing or ameliorating said disorderor disease.

In yet another aspect, the neurological disorder is an increasedanxiety-like response during open field activity testing. In yet anotheraspect, the neurological disorder is a decreased anxiety-like responseduring open field activity testing. In yet another aspect, theneurological disorder is an abnormal circadian rhythm during home-cageactivity testing. In yet another aspect, the neurological disorder is anenhanced motor coordination during inverted screen testing. In yetanother aspect, the neurological disorder is impaired motor coordinationduring inverted screen testing. In yet another aspect, the neurologicaldisorder includes depression, generalized anxiety disorders, attentiondeficit disorder, sleep disorder, hyperactivity disorder, obsessivecompulsive disorder, schizophrenia, cognitive disorders, hyperalgesiaand sensory disorders. Such neurological disorders include the categorydefined as “anxiety disorders” which include but are not limited to:mild to moderate anxiety, anxiety disorder due to a general medicalcondition, anxiety disorder not otherwise specified, generalized anxietydisorder, panic attack, panic disorder with agoraphobia, panic disorderwithout agoraphobia, posttraumatic stress disorder, social phobia,social anxiety, autism, specific phobia, substance-induced anxietydisorder, acute alcohol withdrawal, obsessive compulsive disorder,agoraphobia, monopolar disorders, bipolar disorder I or II, bipolardisorder not otherwise specified, cyclothymic disorder, depressivedisorder, major depressive disorder, mood disorder, substance-inducedmood disorder, enhancement of cognitive function, loss of cognitivefunction associated with but not limited to Alzheimer's disease, stroke,or traumatic injury to the brain, seizures resulting from disease orinjury including but not limited to epilepsy, learningdisorders/disabilities, cerebral palsy. In addition, anxiety disordersmay apply to personality disorders including but not limited to thefollowing types: paranoid, antisocial, avoidant behavior, borderlinepersonality disorders, dependent, histrionic, narcissistic,obsessive-compulsive, schizoid, and schizotypal.

In another aspect, the eye abnormality is a retinal abnormality. Instill another aspect, the eye abnormality is consistent with visionproblems or blindness. In yet another aspect, the retinal abnormality isconsistent with retinitis pigmentosa or is characterized by retinaldegeneration or retinal dysplasia.

In still another aspect, the retinal abnormalities are consistent withretinal dysplasia, various retinopathies, including retinopathy ofprematurity, retrolental fibroplasia, neovascular glaucoma, age-relatedmacular degeneration, diabetic macular edema, cornealneovascularization, corneal graft neovascularization, corneal graftrejection, retinal/choroidal neovascularization, neovascularization ofthe angle (rubeosis), ocular neovascular disease, vascular restenosis,arteriovenous malformations (AVM), meningioma, hemangioma, angiofibroma,thyroid hyperplasias (including Grave's disease), corneal and othertissue transplantation, retinal artery obstruction or occlusion; retinaldegeneration causing secondary atrophy of the retinal vasculature,retinitis pigmentosa, macular dystrophies, Stargardt's disease,congenital stationary night blindness, choroideremia, gyrate atrophy,Leber's congenital amaurosis, retinoschisis disorders, Wagner'ssyndrome, Usher syndromes, Zellweger syndrome, Saldino-Mainzer syndrome,Senior-Loken syndrome, Bardet-Biedl syndrome, Alport's syndrome,Alstrom's syndrome, Cockayne's syndrome, dysplaisa spondyloepiphysariacongentia, Flynn-Aird syndrome, Friedreich ataxia, Hallgren syndrome,Marshall syndrome, Albers-Schnoberg disease, Refsum's disease,Kearns-Sayre syndrome, Waardenburg's syndrome, Alagile syndrome,myotonic dystrophy, olivopontocerebellar atrophy, Pierre-Mariedunsdrome, Stickler syndrome, carotinemeia, cystinosis, Wolframsyndrome, Bassen-Kornzweig syndrome, abetalipoproteinemia, incontinentiapigmenti, Batten's disease, mucopolysaccharidoses, homocystinuria, ormannosidosis.

In still another aspect, the eye abnormality is a cataract. In still yetanother aspect, the cataract is a systemic disease such as human Down'ssyndrome, Hallerman-Streiff syndrome, Lowe syndrome, galactosemia,Marfan syndrome, Trismoy 13-15, Alport syndrome, myotonic dystrophy,Fabry disease, hypoparathroidism or Conradi syndrome.

In still another aspect, the developmental abnormality comprisesembryonic lethality or reduced viability.

In yet another aspect, the cardiovascular, endothelial or angiogenicdisorders are arterial diseases, such as diabetes mellitus; papilledema;optic atrophy; atherosclerosis; angina; myocardial infarctions such asacute myocardial infarctions, cardiac hypertrophy, and heart failuresuch as congestive heart failure; hypertension; inflammatoryvasculitides; Reynaud's disease and Reynaud's phenomenon; aneurysms andarterial restenosis; venous and lymphatic disorders such asthrombophlebitis, lymphangitis, and lymphedema; peripheral vasculardisease; cancer such as vascular tumors, e.g., hemangioma (capillary andcavernous), glomus tumors, telangiectasia, bacillary angiomatosis,hemangioendothelioma, angiosarcoma, haemangiopericytoma, Kaposi'ssarcoma, lymphangioma, and lymphangiosarcoma; tumor angiogenesis; traumasuch as wounds, burns, and other injured tissue, implant fixation,scarring; ischemia reperfusion injury; rheumatoid arthritis;cerebrovascular disease; renal diseases such as acute renal failure, orosteoporosis.

In still yet another aspect, the immunological disorders are consistentwith systemic lupus erythematosis; rheumatoid arthritis; juvenilechronic arthritis; spondyloarthropathies; systemic sclerosis(scleroderma); idiopathic inflammatory myopathies (dermatomyositis,polymyositis); Sjögren's syndrome; systemic vasculitis; sarcoidosis;autoimmune hemolytic anemia (immune pancytopenia, paroxysmal nocturnalhemoglobinuria); autoimmune thrombocytopenia (idiopathicthrombocytopenic purpura, immune-mediated thrombocytopenia); thyroiditis(Grave's disease, Hashimoto's thyroiditis, juvenile lymphocyticthyroiditis, atrophic thyroiditis); diabetes mellitus; immune-mediatedrenal disease (glomerulonephritis, tubulointerstitial nephritis);demyelinating diseases of the central and peripheral nervous systemssuch as multiple sclerosis, idiopathic demyelinating polyneuropathy orGuillain-Barré syndrome, and chronic inflammatory demyelinatingpolyneuropathy; hepatobiliary diseases such as infectious hepatitis(hepatitis A, B, C, D, E and other non-hepatotropic viruses), autoimmunechronic active hepatitis, primary biliary cirrhosis, granulomatoushepatitis, and sclerosing cholangitis; inflammatory bowel disease(ulcerative colitis: Crohn's disease); gluten-sensitive enteropathy, andWhipple's disease; autoimmune or immune-mediated skin diseases includingbullous skin diseases, erythema multiforme and contact dermatitis,psoriasis; allergic diseases such as asthma, allergic rhinitis, atopicdermatitis, food hypersensitivity and urticaria; immunologic diseases ofthe lung such as eosinophilic pneumonia, idiopathic pulmonary fibrosisand hypersensitivity pneumonitis; or transplantation associated diseasesincluding graft rejection and graft-versus-host disease.

In yet another aspect, the bone metabolic abnormality or disorder isarthritis, osteoporosis, osteopenia or osteopetrosis.

In another aspect the therapeutic agent is an agonist or antagonist ofthe phenotype associated with a disruption of a gene which encodes for aPRO226, PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382,PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343,PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571,PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381,PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017,PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196, PRO34778,PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161polypeptide. In yet another aspect, the agent is an agonist orantagonist of a PRO226, PRO257, PRO268, PRO290, PRO36006, PRO363,PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155,PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474,PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348,PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800,PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196,PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161polypeptide. In yet another aspect, the agonist agent is an anti-PRO226,anti-PRO257, anti-PRO268, anti-PRO290, anti-PRO36006, anti-PRO363,anti-PRO365, anti-PRO382, anti-PRO444, anti-PRO705, anti-PRO1071,anti-PRO1125, anti-PRO1134, anti-PRO1155, anti-PRO1281, anti-PRO1343,anti-PRO1379, anti-PRO1380, anti-PRO1387, anti-PRO1419, anti-PRO1433,anti-PRO1474, anti-PRO1550, anti-PRO1571, anti-PRO1572, anti-PRO1759,anti-PRO1904, anti-PRO35193, anti-PRO4341, anti-PRO4348, anti-PRO4369,anti-PRO4381, anti-PRO4407, anti-PRO4425, anti-PRO4985, anti-PRO4989,anti-PRO5737, anti-PRO5800, anti-PRO5993, anti-PRO6017, anti-PRO7174,anti-PRO9744, anti-PRO9821, anti-PRO9852, anti-PRO9873, anti-PRO10196,anti-PRO34778, anti-PRO20233, anti-PRO21956, anti-PRO57290,anti-PRO38465, anti-PRO38683 or anti-PRO85161 antibody. In still another aspect, the antagonist agent is an anti-PRO226, anti-PRO257,anti-PRO268, anti-PRO290, anti-PRO36006, anti-PRO363, anti-PRO365,anti-PRO382, anti-PRO444, anti-PRO705, anti-PRO1071, anti-PRO1125,anti-PRO1134, anti-PRO1155, anti-PRO1281, anti-PRO1343, anti-PRO1379,anti-PRO1380, anti-PRO1387, anti-PRO1419, anti-PRO1433, anti-PRO1474,anti-PRO1550, anti-PRO1571, anti-PRO1572, anti-PRO1759, anti-PRO1904,anti-PRO35193, anti-PRO4341, anti-PRO4348, anti-PRO4369, anti-PRO4381,anti-PRO4407, anti-PRO4425, anti-PRO4985, anti-PRO4989, anti-PRO5737,anti-PRO5800, anti-PRO5993, anti-PRO6017, anti-PRO7174, anti-PRO9744,anti-PRO9821, anti-PRO9852, anti-PRO9873, anti-PRO10196, anti-PRO34778,anti-PRO20233, anti-PRO21956, anti-PRO57290, anti-PRO38465,anti-PRO38683 or anti-PRO85161 antibody.

The invention also provides a method of identifying an agent thatameliorates or modulates a neurological disorder; a cardiovascular,endothelial or angiogenic disorder; an eye abnormality; an immunologicaldisorder; an oncological disorder; a bone metabolic abnormality ordisorder; a lipid metabolic disorder; or a developmental abnormalityassociated with a disruption in the gene which encodes for a PRO226,PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382, PRO444,PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343, PRO1379,PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571, PRO1572,PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381, PRO4407,PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017, PRO7174,PRO9744, PRO9821, PRO9852, PRO9873, PRO10196, PRO34778, PRO20233,PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161 polypeptide, themethod comprising:

(a) providing a non-human transgenic animal cell culture, each cell ofsaid culture comprising a disruption of the gene which encodes for aPRO226, PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382,PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343,PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571,PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381,PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017,PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196, PRO34778,PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161polypeptide;

(b) administering a test agent to said cell culture; and

(c) determining whether the test agent ameliorates or modulates theneurological disorder; cardiovascular, endothelial or angiogenicdisorder; eye abnormality; immunological disorder; oncological disorder;bone metabolic abnormality or disorder; lipid metabolic disorder; ordevelopmental abnormality in said culture. In yet another aspect, theneurological disorder is an increased anxiety-like response during openfield activity testing. In yet another aspect, the neurological disorderis a decreased anxiety-like response during open field activity testing.In yet another aspect, the neurological disorder is an abnormalcircadian rhythm during home-cage activity testing.

In yet another aspect, the neurological disorder is an enhanced motorcoordination during inverted screen testing. In yet another aspect, theneurological disorder is impaired motor coordination during invertedscreen testing. In yet another aspect, the neurological disorderincludes depression, generalized anxiety disorders, attention deficitdisorder, sleep disorder, hyperactivity disorder, obsessive compulsivedisorder, schizophrenia, cognitive disorders, hyperalgesia and sensorydisorders. Such neurological disorders include the category defined as“anxiety disorders” which include but are not limited to: mild tomoderate anxiety, anxiety disorder due to a general medical condition,anxiety disorder not otherwise specified, generalized anxiety disorder,panic attack, panic disorder with agoraphobia, panic disorder withoutagoraphobia, posttraumatic stress disorder, social phobia, socialanxiety, autism, specific phobia, substance-induced anxiety disorder,acute alcohol withdrawal, obsessive compulsive disorder, agoraphobia,monopolar disorders, bipolar disorder I or II, bipolar disorder nototherwise specified, cyclothymic disorder, depressive disorder, majordepressive disorder, mood disorder, substance-induced mood disorder,enhancement of cognitive function, loss of cognitive function associatedwith but not limited to Alzheimer's disease, stroke, or traumatic injuryto the brain, seizures resulting from disease or injury including butnot limited to epilepsy, learning disorders/disabilities, cerebralpalsy. In addition, anxiety disorders may apply to personality disordersincluding but not limited to the following types: paranoid, antisocial,avoidant behavior, borderline personality disorders, dependent,histrionic, narcissistic, obsessive-compulsive, schizoid, andschizotypal.

In another aspect, the eye abnormality is a retinal abnormality. Instill another aspect, the eye abnormality is consistent with visionproblems or blindness. In yet another aspect, the retinal abnormality isconsistent with retinitis pigmentosa or is characterized by retinaldegeneration or retinal dysplasia.

In still another aspect, the retinal abnormalities are consistent withretinal dysplasia, various retinopathies, including retinopathy ofprematurity, retrolental fibroplasia, neovascular glaucoma, age-relatedmacular degeneration, diabetic macular edema, cornealneovascularization, corneal graft neovascularization, corneal graftrejection, retinal/choroidal neovascularization, neovascularization ofthe angle (rubeosis), ocular neovascular disease, vascular restenosis,arteriovenous malformations (AVM), meningioma, hemangioma, angiofibroma,thyroid hyperplasias (including Grave's disease), corneal and othertissue transplantation, retinal artery obstruction or occlusion; retinaldegeneration causing secondary atrophy of the retinal vasculature,retinitis pigmentosa, macular dystrophies, Stargardt's disease,congenital stationary night blindness, choroideremia, gyrate atrophy,Leber's congenital amaurosis, retinoschisis disorders, Wagner'ssyndrome, Usher syndromes, Zellweger syndrome, Saldino-Mainzer syndrome,Senior-Loken syndrome, Bardet-Biedl syndrome, Alport's syndrome,Alstrom's syndrome, Cockayne's syndrome, dysplaisa spondyloepiphysariacongentia, Flynn-Aird syndrome, Friedreich ataxia, Hallgren syndrome,Marshall syndrome, Albers-Schnoberg disease, Refsum's disease,Kearns-Sayre syndrome, Waardenburg's syndrome, Alagile syndrome,myotonic dystrophy, olivopontocerebellar atrophy, Pierre-Mariedunsdrome, Stickler syndrome, carotinemeia, cystinosis, Wolframsyndrome, Bassen-Kornzweig syndrome, abetalipoproteinemia, incontinentiapigmenti, Batten's disease, mucopolysaccharidoses, homocystinuria, ormannosidosis.

In still another aspect, the eye abnormality is a cataract. In still yetanother aspect, the cataract is a systemic disease such as human Down'ssyndrome, Hallerman-Streiff syndrome, Lowe syndrome, galactosemia,Marfan syndrome, Trismoy 13-15, Alport syndrome, myotonic dystrophy,Fabry disease, hypoparathroidism or Conradi syndrome.

In still another aspect, the developmental abnormality comprisesembryonic lethality or reduced viability.

In yet another aspect, the cardiovascular, endothelial or angiogenicdisorders are arterial diseases, such as diabetes mellitus; papilledema;optic atrophy; atherosclerosis; angina; myocardial infarctions such asacute myocardial infarctions, cardiac hypertrophy, and heart failuresuch as congestive heart failure; hypertension; inflammatoryvasculitides; Reynaud's disease and Reynaud's phenomenon; aneurysms andarterial restenosis; venous and lymphatic disorders such asthrombophlebitis, lymphangitis, and lymphedema; peripheral vasculardisease; cancer such as vascular tumors, e.g., hemangioma (capillary andcavernous), glomus tumors, telangiectasia, bacillary angiomatosis,hemangioendothelioma, angiosarcoma, haemangiopericytoma, Kaposi'ssarcoma, lymphangioma, and lymphangiosarcoma; tumor angiogenesis; traumasuch as wounds, burns, and other injured tissue, implant fixation,scarring; ischemia reperfusion injury; rheumatoid arthritis;cerebrovascular disease; renal diseases such as acute renal failure, orosteoporosis.

In still yet another aspect, the immunological disorders are consistentwith systemic lupus erythematosis; rheumatoid arthritis; juvenilechronic arthritis; spondyloarthropathies; systemic sclerosis(scleroderma); idiopathic inflammatory myopathies (dermatomyositis,polymyositis); Sjögren's syndrome; systemic vasculitis; sarcoidosis;autoimmune hemolytic anemia (immune pancytopenia, paroxysmal nocturnalhemoglobinuria); autoimmune thrombocytopenia (idiopathicthrombocytopenic purpura, immune-mediated thrombocytopenia); thyroiditis(Grave's disease, Hashimoto's thyroiditis, juvenile lymphocyticthyroiditis, atrophic thyroiditis); diabetes mellitus; immune-mediatedrenal disease (glomerulonephritis, tubulointerstitial nephritis);demyelinating diseases of the central and peripheral nervous systemssuch as multiple sclerosis, idiopathic demyelinating polyneuropathy orGuillain-Barré syndrome, and chronic inflammatory demyelinatingpolyneuropathy; hepatobiliary diseases such as infectious hepatitis(hepatitis A, B, C, D, E and other non-hepatotropic viruses), autoimmunechronic active hepatitis, primary biliary cirrhosis, granulomatoushepatitis, and sclerosing cholangitis; inflammatory bowel disease(ulcerative colitis: Crohn's disease); gluten-sensitive enteropathy, andWhipple's disease; autoimmune or immune-mediated skin diseases includingbullous skin diseases, erythema multiforme and contact dermatitis,psoriasis; allergic diseases such as asthma, allergic rhinitis, atopicdermatitis, food hypersensitivity and urticaria; immunologic diseases ofthe lung such as eosinophilic pneumonia, idiopathic pulmonary fibrosisand hypersensitivity pneumonitis; or transplantation associated diseasesincluding graft rejection and graft-versus-host disease.

In yet another aspect, the bone metabolic abnormality or disorder isarthritis, osteoporosis, osteopenia or osteopetrosis.

The invention also provides an agent that ameliorates or modulates aneurological disorder; a cardiovascular, endothelial or angiogenicdisorder; an eye abnormality; an immunological disorder; an oncologicaldisorder; a bone metabolic abnormality or disorder; a lipid metabolicdisorder; or a developmental abnormality which is associated with genedisruption in said culture. In one aspect, the agent is an agonist orantagonist of the phenotype associated with a disruption of a gene whichencodes for a PRO226, PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365,PRO382, PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281,PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550,PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369,PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993,PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196,PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161polypeptide. In yet another aspect, the agent is an agonist or antagonist of a PRO226, PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365,PRO382, PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281,PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550,PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369,PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993,PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196,PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161polypeptide. In yet another aspect, the agonist agent is an anti-PRO226,anti-PRO257, anti-PRO268, anti-PRO290, anti-PRO36006, anti-PRO363,anti-PRO365, anti-PRO382, anti-PRO444, anti-PRO705, anti-PRO1071,anti-PRO1125, anti-PRO1134, anti-PRO1155, anti-PRO1281, anti-PRO1343,anti-PRO1379, anti-PRO1380, anti-PRO1387, anti-PRO1419, anti-PRO1433,anti-PRO1474, anti-PRO1550, anti-PRO1571, anti-PRO1572, anti-PRO1759,anti-PRO1904, anti-PRO35193, anti-PRO4341, anti-PRO4348, anti-PRO4369,anti-PRO4381, anti-PRO4407, anti-PRO4425, anti-PRO4985, anti-PRO4989,anti-PRO5737, anti-PRO5800, anti-PRO5993, anti-PRO6017, anti-PRO7174,anti-PRO9744, anti-PRO9821, anti-PRO9852, anti-PRO9873, anti-PRO10196,anti-PRO34778, anti-PRO20233, an, anti-PRO21956, anti-PRO57290,anti-PRO38465, anti-PRO38683 or anti-PRO85161 antibody. In still anotheraspect, the antagonist agent is an anti-PRO226, anti-PRO257,anti-PRO268, anti-PRO290, anti-PRO36006, anti-PRO363, anti-PRO365,anti-PRO382, anti-PRO444, anti-PRO705, anti-PRO1071, anti-PRO1125,anti-PRO1134, anti-PRO1155, anti-PRO1281, anti-PRO1343, anti-PRO1379,anti-PRO1380, anti-PRO1387, anti-PRO1419, anti-PRO1433, anti-PRO1474,anti-PRO1550, anti-PRO1571, anti-PRO1572, anti-PRO1759, anti-PRO1904,anti-PRO35193, anti-PRO4341, anti-PRO4348, anti-PRO4369, anti-PRO4381,anti-PRO4407, anti-PRO4425, anti-PRO4985, anti-PRO4989, anti-PRO5737,anti-PRO5800, anti-PRO5993, anti-PRO6017, anti-PRO7174, anti-PRO9744,anti-PRO9821, anti-PRO9852, anti-PRO9873, anti-PRO10196, anti-PRO34778,anti-PRO20233, anti-PRO21956, anti-PRO57290, anti-PRO38465,anti-PRO38683 or anti-PRO85161 antibody.

The invention also provides a method of modulating a phenotypeassociated with a disruption of a gene which encodes for a PRO226,PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382, PRO444,PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343, PRO1379,PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571, PRO1572,PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381, PRO4407,PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017, PRO7174,PRO9744, PRO9821, PRO9852, PRO9873, PRO10196, PRO34778, PRO20233,PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161 polypeptide, themethod comprising administering to a subject whom may already have thephenotype, or may be prone to have the phenotype or may be in whom thephenotype is to be prevented, an effective amount of an agent identifiedas modulating said phenotype, or agonists or antagonists thereof,thereby effectively modulating the phenotype.

The invention also provides a method of modulating a physiologicalcharacteristic associated with a disruption of a gene which encodes fora PRO226, PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382,PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343,PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571,PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381,PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017,PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196, PRO34778,PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161polypeptide, the method comprising administering to a subject whom mayalready exhibit the physiological characteristic, or may be prone toexhibit the physiological characteristic or may be in whom thephysiological characteristic is to be prevented, an effective amount ofan agent identified as modulating said physiological characteristic, oragonists or antagonists thereof, thereby effectively modulating thephysiological characteristic.

The invention also provides a method of modulating a behavior associatedwith a disruption of a gene which encodes for a PRO226, PRO257, PRO268,PRO290, PRO36006, PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071,PRO1125, PRO1134, PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387,PRO1419, PRO1433, PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904,PRO35193, PRO4341, PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985,PRO4989, PRO5737, PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821,PRO9852, PRO9873, PRO10196, PRO34778, PRO20233, PRO21956, PRO57290,PRO38465, PRO38683 or PRO85161 polypeptide, the method comprisingadministering to a subject whom may already exhibit the behavior, or maybe prone to exhibit the behavior or may be in whom the exhibitedbehavior is to be prevented, an effective amount of an agent identifiedas modulating said behavior, or agonists or antagonists thereof, therebyeffectively modulating the behavior.

The invention also provides a method of modulating the expression of aPRO226, PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382,PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343,PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571,PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381,PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017,PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196, PRO34778,PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161polypeptide, the method comprising administering to a host cellexpressing said PRO226, PRO257, PRO268, PRO290, PRO36006, PRO363,PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125, PRO134, PRO1155,PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474,PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348,PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800,PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196,PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161polypeptide, an effective amount of an agent identified as modulatingsaid expression, or agonists or antagonists thereof, thereby effectivelymodulating the expression of said polypeptide.

The invention also provides a method of modulating a conditionassociated with a disruption of a gene which encodes for a PRO226,PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382, PRO444,PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343, PRO1379,PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571, PRO1572,PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381, PRO4407,PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017, PRO7174,PRO9744, PRO9821, PRO9852, PRO9873, PRO10196, PRO34778, PRO20233,PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161 polypeptide, themethod comprising administering to a subject whom may have thecondition, or may be prone to have the condition or may be in whom thecondition is to be prevented, a therapeutically effective amount of atherapeutic agent identified as modulating said condition, or agonistsor antagonists thereof, thereby effectively modulating the condition.

The invention also provides a method of treating or preventing orameliorating a neurological disorder; cardiovascular, endothelial orangiogenic disorder; immunological disorder; oncological disorder; bonemetabolic abnormality or disorder, or embryonic lethality associatedwith the disruption of a gene which encodes for a PRO226, PRO257,PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382, PRO444, PRO705,PRO171, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343, PRO1379, PRO1380,PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571, PRO1572, PRO1759,PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381, PRO4407, PRO4425,PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017, PRO7174, PRO9744,PRO9821, PRO9852, PRO9873, PRO10196, PRO34778, PRO20233, PRO21956,PRO57290, PRO38465, PRO38683 or PRO85161 polypeptide, the methodcomprising administering to a non-human transgenic animal cell culture,each cell of said culture comprising a disruption of the gene whichencodes for a PRO226, PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365,PRO382, PRO444, PRO705, PRO171, PRO1125, PRO1134, PRO1155, PRO1281,PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550,PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369,PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993,PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196,PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161polypeptide, an effective amount of an agent identified as treating orpreventing or ameliorating said disorder, or agonists or antagoniststhereof, thereby effectively treating or preventing or ameliorating saiddisorder.

B. Further Embodiments

In yet further embodiments, the invention is directed to the followingset of potential claims for this application:

1. A method of identifying a phenotype associated with a disruption of agene which encodes for a PRO226, PRO257, PRO268, PRO290, PRO36006,PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125, PRO1134,PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433,PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341,PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737,PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873,PRO10196, PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 orPRO85161 polypeptide, the method comprising:

(a) providing a non-human transgenic animal whose genome comprises adisruption of the gene which encodes for a PRO226, PRO257, PRO268,PRO290, PRO36006, PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071,PRO1125, PRO1134, PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387,PRO1419, PRO1433, PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904,PRO35193, PRO4341, PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985,PRO4989, PRO5737, PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821,PRO9852, PRO9873, PRO10196, PRO34778, PRO20233, PRO21956, PRO57290,PRO38465, PRO38683 or PRO85161 polypeptide;

(b) measuring a physiological characteristic of the non-human transgenicanimal; and

(c) comparing the measured physiological characteristic with that of agender matched wild-type animal, wherein the physiologicalcharacteristic of the non-human transgenic animal that differs from thephysiological characteristic of the wild-type animal is identified as aphenotype resulting from the gene disruption in the non-human transgenicanimal.

2. The method of Claim 1, wherein the non-human transgenic animal isheterozygous for the disruption of a gene which encodes for a PRO226,PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382, PRO444,PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343, PRO1379,PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571, PRO1572,PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381, PRO4407,PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017, PRO7174,PRO9744, PRO9821, PRO9852, PRO9873, PRO10196, PRO34778, PRO20233,PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161 polypeptide.3. The method of Claim 1, wherein the phenotype exhibited by thenon-human transgenic animal as compared with gender matched wild-typelittermates is at least one of the following: a neurological disorder; acardiovascular, endothelial or angiogenic disorder; an eye abnormality;an immunological disorder; an oncological disorder; a bone metabolicabnormality or disorder; a lipid metabolic disorder; or a developmentalabnormality.4. The method of Claim 3, wherein the neurological disorder is anincreased anxiety-like response during open field activity testing.5. The method of Claim 3, wherein the neurological disorder is adecreased anxiety-like response during open field activity testing.6. The method of Claim 3, wherein the neurological disorder is anabnormal circadian rhythm during home-cage activity testing.7. The method of Claim 3, wherein the neurological disorder is anenhanced motor coordination during inverted screen testing.8. The method of Claim 3, wherein the neurological disorder is animpaired motor coordination during inverted screen testing.9. The method of Claim 3, wherein the neurological disorder isdepression, generalized anxiety disorders, attention deficit disorder,sleep disorder, hyperactivity disorder, obsessive compulsive disorder,schizophrenia, cognitive disorders, hyperalgesia or sensory disorders.10. The method of Claim 3, wherein the eye abnormality is a retinalabnormality.11. The method of Claim 3, wherein the eye abnormality is consistentwith vision problems or blindness.12. The method of Claim 10, wherein the retinal abnormality isconsistent with retinitis pigmentosa.13. The method of Claim 10, wherein the retinal abnormality ischaracterized by retinal degeneration or retinal dysplasia.14. The method of Claim 10, wherein the retinal abnormality isconsistent with retinal dysplasia, various retinopathies, includingretinopathy of prematurity, retrolental fibroplasia, neovascularglaucoma, age-related macular degeneration, diabetic macular edema,corneal neovascularization, corneal graft neovascularization, cornealgraft rejection, retinal/choroidal neovascularization,neovascularization of the angle (rubeosis), ocular neovascular disease,vascular restenosis, arteriovenous malformations (AVM), meningioma,hemangioma, angiofibroma, thyroid hyperplasias (including Grave'sdisease), corneal and other tissue transplantation, retinal arteryobstruction or occlusion; retinal degeneration causing secondary atrophyof the retinal vasculature, retinitis pigmentosa, macular dystrophies,Stargardt's disease, congenital stationary night blindness,choroideremia, gyrate atrophy, Leber's congenital amaurosis,retinoschisis disorders, Wagner's syndrome, Usher syndromes, Zellwegersyndrome, Saldino-Mainzer syndrome, Senior-Loken syndrome, Bardet-Biedlsyndrome, Alport's syndrome, Alstrom's syndrome, Cockayne's syndrome,dysplaisa spondyloepiphysaria congentia, Flynn-Aird syndrome, Friedreichataxia, Hallgren syndrome, Marshall syndrome, Albers-Schnoberg disease,Refsum's disease, Kearns-Sayre syndrome, Waardenburg's syndrome, Alagilesyndrome, myotonic dystrophy, olivopontocerebellar atrophy, Pierre-Mariedunsdrome, Stickler syndrome, carotinemeia, cystinosis, Wolframsyndrome, Bassen-Kornzweig syndrome, abetalipoproteinemia, incontinentiapigmenti, Batten's disease, mucopolysaccharidoses, homocystinuria, ormannosidosis.15. The method of Claim 3, wherein the eye abnormality is a cataract.16. The method of Claim 15, wherein the cataract is consistent withsystemic diseases such as human Down's syndrome, Hallerman-Streiffsyndrome, Lowe syndrome, galactosemia, Marfan syndrome, Trismoy 13-15,Alport syndrome, myotonic dystrophy, Fabry disease, hypoparathroidism orConradi syndrome.17. The method of Claim 3, wherein the developmental abnormalitycomprises embryonic lethality or reduced viability.18. The method of Claim 3, wherein the cardiovascular, endothelial orangiogenic disorders are arterial diseases, such as diabetes mellitus;papilledema; optic atrophy; atherosclerosis; angina; myocardialinfarctions such as acute myocardial infarctions, cardiac hypertrophy,and heart failure such as congestive heart failure; hypertension;inflammatory vasculitides; Reynaud's disease and Reynaud's phenomenon;aneurysms and arterial restenosis; venous and lymphatic disorders suchas thrombophlebitis, lymphangitis, and lymphedema; peripheral vasculardisease; cancer such as vascular tumors, e.g., hemangioma (capillary andcavernous), glomus tumors, telangiectasia, bacillary angiomatosis,hemangioendothelioma, angiosarcoma, haemangiopericytoma, Kaposi'ssarcoma, lymphangioma, and lymphangiosarcoma; tumor angiogenesis; traumasuch as wounds, burns, and other injured tissue, implant fixation,scarring; ischemia reperfusion injury; rheumatoid arthritis;cerebrovascular disease; renal diseases such as acute renal failure, orosteoporosis.19. The method of Claim 3, wherein the immunological disorders aresystemic lupus erythematosis; rheumatoid arthritis; juvenile chronicarthritis; spondyloarthropathies; systemic sclerosis (scleroderma);idiopathic inflammatory myopathies (dermatomyositis, polymyositis);Sjögren's syndrome; systemic vasculitis; sarcoidosis; autoimmunehemolytic anemia (immune pancytopenia, paroxysmal nocturnalhemoglobinuria); autoimmune thrombocytopenia (idiopathicthrombocytopenic purpura, immune-mediated thrombocytopenia); thyroiditis(Grave's disease, Hashimoto's thyroiditis, juvenile lymphocyticthyroiditis, atrophic thyroiditis); diabetes mellitus; immune-mediatedrenal disease (glomerulonephritis, tubulointerstitial nephritis);demyelinating diseases of the central and peripheral nervous systemssuch as multiple sclerosis, idiopathic demyelinating polyneuropathy orGuillain-Barré syndrome, and chronic inflammatory demyelinatingpolyneuropathy; hepatobiliary diseases such as infectious hepatitis(hepatitis A, B, C, D, E and other non-hepatotropic viruses), autoimmunechronic active hepatitis, primary biliary cirrhosis, granulomatoushepatitis, and sclerosing cholangitis; inflammatory bowel disease(ulcerative colitis: Crohn's disease); gluten-sensitive enteropathy, andWhipple's disease; autoimmune or immune-mediated skin diseases includingbullous skin diseases, erythema multiforme and contact dermatitis,psoriasis; allergic diseases such as asthma, allergic rhinitis, atopicdermatitis, food hypersensitivity and urticaria; immunologic diseases ofthe lung such as eosinophilic pneumonia, idiopathic pulmonary fibrosisand hypersensitivity pneumonitis; or transplantation associated diseasesincluding graft rejection and graft-versus-host disease.20. The method of Claim 3, wherein the bone metabolic abnormality ordisorder is arthritis, osteoporosis or osteopetrosis.21. The method of Claim 1, wherein the non-human transgenic animalexhibits at least one of the following physiological characteristicscompared with gender matched wild-type littermates: increasedanxiety-like response during open field testing; decreased anxietyduring open field testing; decreased locomotor activity during openfield testing; abnormal circadian rhythm during home-cage activitytesting (low activity during the light phase); abnormal circadian rhythmduring home-cage activity testing including decreased ambulatory counts;hypoactivity with no circadian rhythm; abnormal circadian rhythm duringhome-cage activity testing including increased ambulatory counts;increased stress induced hyperthermia; decreased stress inducedhyperthermia; impaired motor coordination during inverted screentesting; increased immobility in tail suspension (increaseddepressive-like response); increased depressive-like response duringtail suspension testing; increased immobility or decreaseddepressive-like response during tail suspension testing; decreasedstartle response during prepulse inhibition testing; no startle responseindicating deafness; or impaired hearing; decreased prepulse inhibitionwith impaired sensorimotor gating/attention; decreased responsiveness inhotplate testing; decreased latency to respond in hot plate testing;opthamological abnormalities; increased mean artery-to-vein ratio;resistance to pupil dilating drug cyclopentolate hydrochloride; squintyeyes; squint eyes with white spots; cataracts; retinal degeneration;impaired vision; decreased basal body temperature; decreased heart rate;increased mean systolic blood pressure; increased insulin sensitivity;increased mean fasting serum glucose levels; decreased mean serumglucose levels; increased mean serum cholesterol levels; decreased meanserum cholesterol levels; increased mean serum triglyceride levels;decreased mean serum triglyceride levels; enhanced glucose tolerance;impaired glucose tolerance; decreased mean serum insulin levels;increased mean serum calcium; increased urobilinogen, notable lipemia;increased albumin, alanine amino transferase, phosphorus and potassiumlevels; increased mean serum alkaline phosphatase levels; increasedblood urea nitrogen; increased percentage of granulocyte; increasedtotal white blood cell (WBC) count; increased mean absolute neutrophilcount; neutropenia; increased absolute lymphocyte count; increasedabsolute monocyte count; increased monocytes and DC in spleen (CD11b+,CD11b+c+); increased mean platelet count; increased natural killer (NK)cells in lymph node; decreased neutrophil count; decreased naturalkiller (NK) cells; decreased mean red blood cell (RBC) count, hemoglobinconcentration, and hematocrit; increased mean red cell distributionwidth; decreased mean corpuscular volume and mean corpuscularhemoglobin; decreased mean platelet count and increased platelet volume;increase B cell number in lymph node; increase in B cell subtypes inPeyer's patches; increased percentage of B cells in lymph node; increaseCD25+ cells; increased thymic DN, decreased DP T cells; increased CD19+cells in lymph node; increased CD117 in bone marrow cells; increasedmean percentage of CD4 cells; increased CD8 cells and decrease in Bcells; increased percentage CD11b+ cells in peritoneal lavage; increasedpercentage of B220+ CD11b Low CD23− cells; increased percentages ofB220− CD11 Low and CD11b− cells in peritoneal lavage; increasedpercentage of B220−CD11bHi cells in peritoneal lavage; decreasedpercentage of B220+ CD11b− CD23+ cells in peritoneal lavage; increasedpercentage of B220− CD43 Hi cells in bone marrow; increased CD11b+CD11c−cells in spleen; increase in CD62hi, CD44int subsets of CD4 and CD8cells; increase in peritoneal CD117 cells; increase TcRbeta/CD38 cellsin Peyer's patches; increased percentage of TcRbeta+ cells in thymus;increased percentages of CD11b+CD11c+ in lymph node; decreasedpercentage of B220+ Hi CD23+ cells in peritoneal lavage; decreasedpercentage of B220+ Med CD23−cells in peritoneal lavage; decreasedpercentages of CD62L Hi CD44 Dim CD4+ and CD8+ cells in spleen;decreased percentage of B220−CD11b Hi cells; decreased mean percentagesof CD4 and CD8 cells in lymph node and spleen; increased memory T cells[increased CD62L lo CD44hi]; decreased T cell:B cell ratio; decreasednaive T cells; decreased CD117 cells in peritoneal lavage; decreasedmean percentage of CD8 cells, increased IgG1 response to ovalbuminchallenge; increased IgG2a response to ovalbumin challenge; increasedmean serum IL-6 response to LPS challenge; increased TNF alpha responseto LPS challenge; increased serum MCP-1 response to LPS challenge;increased mean serum IgM level; increased serum IgA; increase mean serumIgG1; increased mean serum IgG3; decreased serum IgG1 response toovalbumin challenge; decreased serum IgG2a response to ovalbuminchallenge; decreased mean serum IgA level; decreased serum IgG2a level;decrease in serum IgG3 level; increased skin fibroblast proliferationrate; decreased skin fibroblast proliferation rate; increased meanpercent of total body fat and total fat mass; increased mean bodyweight; increased mean body length; increased total tissue mass (TTM);increased mean femoral midshaft cortical thickness and cross-sectionalarea; increased mean vertebral trabecular bone volume, number andconnectivity density; decreased mean percent of total body fat and totalfat mass; decreased mean body weight; decreased mean body length;decreased total tissue mass (TTM); decreased lean body mass (LBM);decreased femoral bone mineral density (BMD); decreased vertebral bonemineral density (BMD); decreased bone mineral density (BMD) in totalbody, femur and vertebrae; decreased bone mineral content (BMC);decreased bone mineral density index; decreased volumetric bone mineraldensity (vBMD); decreased mean femoral midshaft cortical thickness andcross-sectional area; decreased mean vertebral trabecular bone volume,number and connectivity density; osteopetrosis; osteoporosis; chronicinflammation in various tissues; bilateral hydronephrosis (moderate tosevere) and inflammation; “pear shaped abdomen”; bilaterally enlargedkidneys, suggesting polycystic kidney disease; degeneration of the Organof Corti; hepatocellular dysfunction; biliary obstruction;hepatosplenomegaly characterized by histiocytic infiltrate;histiocytosis in the small intestine, lymph nodes and spleen;splenomegaly, lymphadenopathy and lymphadenopathy; hyperplasia ofadenoid and tonsils; mild-moderate extra medullary hematopoiesis;homozygous mice were small, dehydrated and exhibited decreasedsubcutaneous fat depots; lipopenia; ulcerous colitis; diffuse markeddegeneration of sensory cochlear hair cells in the inner ear,characterized by a complete loss of both inner and outer cochlear haircells on the basilar membrane; gastric mucosal hyperplasia and chronicinflammation; in creased stomach weight; defective spermatogensis in thetestes; hypospermia and defective spermatozoa in the epididymus; maleinfertility; lysosomal storage disease; anemia; growth retardation;reduced viability; perinatal lethality with decreased lymphocytes andlipopenia; homozygous embryonic lethality; and heterozygous embryoniclethality.22. An isolated cell derived from a non-human transgenic animal whosegenome comprises a disruption of the gene which encodes for a PRO226,PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382, PRO444,PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343, PRO1379,PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571, PRO1572,PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381, PRO4407,PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017, PRO7174,PRO9744, PRO9821, PRO9852, PRO9873, PRO10196, PRO34778, PRO20233,PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161 polypeptide.23. The isolated cell of Claim 22 which is a murine cell.24. The isolated cell of Claim 23, wherein the murine cell is anembryonic stem cell.25. The isolated cell of Claim 22, wherein the non-human transgenicanimal exhibits at least one of the following phenotypes compared withgender matched wild-type littermates: a neurological disorder; acardiovascular, endothelial or angiogenic disorder; an eye abnormality;an immunological disorder; an oncological disorder; a bone metabolicabnormality or disorder; a lipid metabolic disorder; or a developmentalabnormality.26. A method of identifying an agent that modulates a phenotypeassociated with a disruption of a gene which encodes for a PRO226,PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382, PRO444,PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343, PRO1379,PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571, PRO1572,PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381, PRO4407,PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017, PRO7174,PRO9744, PRO9821, PRO9852, PRO9873, PRO10196, PRO34778, PRO20233,PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161 polypeptide, themethod comprising:

(a) providing a non-human transgenic animal whose genome comprises adisruption of the gene which encodes for the PRO226, PRO257, PRO268,PRO290, PRO36006, PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071,PRO1125, PRO1134, PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387,PRO1419, PRO1433, PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904,PRO35193, PRO4341, PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985,PRO4989, PRO5737, PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821,PRO9852, PRO9873, PRO10196, PRO34778, PRO20233, PRO21956, PRO57290,PRO38465, PRO38683 or PRO85161 polypeptide;

(b) measuring a physiological characteristic of the non-human transgenicanimal of (a);

(c) comparing the measured physiological characteristic of (b) with thatof a gender matched wild-type animal, wherein the physiologicalcharacteristic of the non-human transgenic animal that differs from thephysiological characteristic of the wild-type animal is identified as aphenotype resulting from the gene disruption in the non-human transgenicanimal;

(d) administering a test agent to the non-human transgenic animal of(a); and

(e) determining whether the test agent modulates the identifiedphenotype associated with gene disruption in the non-human transgenicanimal.

27. The method of Claim 26, wherein the phenotype associated with thegene disruption comprises a neurological disorder; a cardiovascular,endothelial or angiogenic disorder; an eye abnormality; an immunologicaldisorder; an oncological disorder; a bone metabolic abnormality ordisorder; a lipid metabolic disorder; or a developmental abnormality.28. The method of Claim 27, wherein the neurological disorder is anincreased anxiety-like response during open field activity testing.29. The method of Claim 27, wherein the neurological disorder is adecreased anxiety-like response during open field activity testing.30. The method of Claim 27, wherein the neurological disorder is anabnormal circadian rhythm during home-cage activity testing.31. The method of Claim 27, wherein the neurological disorder is anenhanced motor coordination during inverted screen testing.32. The method of Claim 27, wherein the neurological disorder is animpaired motor coordination during inverted screen testing.33. The method of Claim 27, wherein the neurological disorder isdepression, generalized anxiety disorders, attention deficit disorder,sleep disorder, hyperactivity disorder, obsessive compulsive disorder,schizophrenia, cognitive disorders, hyperalgesia or sensory disorders.34. The method of Claim 27, wherein the eye abnormality is a retinalabnormality.35. The method of Claim 27, wherein the eye abnormality is consistentwith vision problems or blindness.36. The method of Claim 34, wherein the retinal abnormality isconsistent with retinitis pigmentosa.37. The method of Claim 34, wherein the retinal abnormality ischaracterized by retinal degeneration or retinal dysplasia.38. The method of Claim 34, wherein the retinal abnormality isconsistent with retinal dysplasia, various retinopathies, includingretinopathy of prematurity, retrolental fibroplasia, neovascularglaucoma, age-related macular degeneration, diabetic macular edema,corneal neovascularization, corneal graft neovascularization, cornealgraft rejection, retinal/choroidal neovascularization,neovascularization of the angle (rubeosis), ocular neovascular disease,vascular restenosis, arteriovenous malformations (AVM), meningioma,hemangioma, angiofibroma, thyroid hyperplasias (including Grave'sdisease), corneal and other tissue transplantation, retinal arteryobstruction or occlusion; retinal degeneration causing secondary atrophyof the retinal vasculature, retinitis pigmentosa, macular dystrophies,Stargardt's disease, congenital stationary night blindness,choroideremia, gyrate atrophy, Leber's congenital amaurosis,retinoschisis disorders, Wagner's syndrome, Usher syndromes, Zellwegersyndrome, Saldino-Mainzer syndrome, Senior-Loken syndrome, Bardet-Biedlsyndrome, Alport's syndrome, Alstrom's syndrome, Cockayne's syndrome,dysplaisa spondyloepiphysaria congentia, Flynn-Aird syndrome, Friedreichataxia, Hallgren syndrome, Marshall syndrome, Albers-Schnoberg disease,Refsum's disease, Kearns-Sayre syndrome, Waardenburg's syndrome, Alagilesyndrome, myotonic dystrophy, olivopontocerebellar atrophy, Pierre-Mariedunsdrome, Stickler syndrome, carotinemeia, cystinosis, Wolframsyndrome, Bassen-Kornzweig syndrome, abetalipoproteinemia, incontinentiapigmenti, Batten's disease, mucopolysaccharidoses, homocystinuria, ormannosidosis.39. The method of Claim 27, wherein the eye abnormality is a cataract.40. The method of Claim 39, wherein the cataract is consistent withsystemic diseases such as human Down's syndrome, Hallerman-Streiffsyndrome, Lowe syndrome, galactosemia, Marfan syndrome, Trismoy 13-15,Alport syndrome, myotonic dystrophy, Fabry disease, hypoparathroidism orConradi syndrome.41. The method of Claim 27, wherein the developmental abnormalitycomprises embryonic lethality or reduced viability.42. The method of Claim 27, wherein the cardiovascular, endothelial orangiogenic disorders are arterial diseases, such as diabetes mellitus;papilledema; optic atrophy; atherosclerosis; angina; myocardialinfarctions such as acute myocardial infarctions, cardiac hypertrophy,and heart failure such as congestive heart failure; hypertension;inflammatory vasculitides; Reynaud's disease and Reynaud's phenomenon;aneurysms and arterial restenosis; venous and lymphatic disorders suchas thrombophlebitis, lymphangitis, and lymphedema; peripheral vasculardisease; cancer such as vascular tumors, e.g., hemangioma (capillary andcavernous), glomus tumors, telangiectasia, bacillary angiomatosis,hemangioendothelioma, angiosarcoma, haemangiopericytoma, Kaposi'ssarcoma, lymphangioma, and lymphangiosarcoma; tumor angiogenesis; traumasuch as wounds, burns, and other injured tissue, implant fixation,scarring; ischemia reperfusion injury; rheumatoid arthritis;cerebrovascular disease; renal diseases such as acute renal failure, orosteoporosis.43. The method of Claim 27, wherein the immunological disorders aresystemic lupus erythematosis; rheumatoid arthritis; juvenile chronicarthritis; spondyloarthropathies; systemic sclerosis (scleroderma);idiopathic inflammatory myopathies (dermatomyositis, polymyositis);Sjögren's syndrome; systemic vasculitis; sarcoidosis; autoimmunehemolytic anemia (immune pancytopenia, paroxysmal nocturnalhemoglobinuria); autoimmune thrombocytopenia (idiopathicthrombocytopenic purpura, immune-mediated thrombocytopenia); thyroiditis(Grave's disease, Hashimoto's thyroiditis, juvenile lymphocyticthyroiditis, atrophic thyroiditis); diabetes mellitus; immune-mediatedrenal disease (glomerulonephritis, tubulointerstitial nephritis);demyelinating diseases of the central and peripheral nervous systemssuch as multiple sclerosis, idiopathic demyelinating polyneuropathy orGuillain-Barré syndrome, and chronic inflammatory demyelinatingpolyneuropathy; hepatobiliary diseases such as infectious hepatitis(hepatitis A, B, C, D, E and other non-hepatotropic viruses), autoimmunechronic active hepatitis, primary biliary cirrhosis, granulomatoushepatitis, and sclerosing cholangitis; inflammatory bowel disease(ulcerative colitis: Crohn's disease); gluten-sensitive enteropathy, andWhipple's disease; autoimmune or immune-mediated skin diseases includingbullous skin diseases, erythema multiforme and contact dermatitis,psoriasis; allergic diseases such as asthma, allergic rhinitis, atopicdermatitis, food hypersensitivity and urticaria; immunologic diseases ofthe lung such as eosinophilic pneumonia, idiopathic pulmonary fibrosisand hypersensitivity pneumonitis; or transplantation-associated diseasesincluding graft rejection and graft-versus-host disease.44. The method of Claim 27, wherein said bone metabolic abnormality ordisorder is arthritis, osteoporosis or osteopetrosis.45. The method of Claim 26, wherein the non-human transgenic animalexhibits at least one of the following physiological characteristicscompared with gender matched wild-type littermates: increasedanxiety-like response during open field testing; decreased anxietyduring open field testing; decreased locomotor activity during openfield testing; abnormal circadian rhythm during home-cage activitytesting (low activity during the light phase); abnormal circadian rhythmduring home-cage activity testing including decreased ambulatory counts;hypoactivity with no circadian rhythm; abnormal circadian rhythm duringhome-cage activity testing including increased ambulatory counts;increased stress induced hyperthermia; decreased stress inducedhyperthermia; impaired motor coordination during inverted screentesting; increased immobility in tail suspension (increaseddepressive-like response); increased depressive-like response duringtail suspension testing; increased immobility or decreaseddepressive-like response during tail suspension testing; decreasedstartle response during prepulse inhibition testing; no startle responseindicating deafness; or impaired hearing; decreased prepulse inhibitionwith impaired sensorimotor gating/attention; decreased responsiveness inhot plate testing; decreased latency to respond in hot plate testing;opthamological abnormalities; increased mean artery-to-vein ratio;resistance to pupil dilating drug cyclopentolate hydrochloride; squintyeyes; squint eyes with white spots; cataracts; retinal degeneration;impaired vision; decreased basal body temperature; decreased heart rate;increased mean systolic blood pressure; increased insulin sensitivity;increased mean fasting serum glucose levels; decreased mean serumglucose levels; increased mean serum cholesterol levels; decreased meanserum cholesterol levels; increased mean serum triglyceride levels;decreased mean serum triglyceride levels; enhanced glucose tolerance;impaired glucose tolerance; decreased mean serum insulin levels;increased mean serum calcium; increased urobilinogen, notable lipemia;increased albumin, alanine amino transferase, phosphorus and potassiumlevels; increased mean serum alkaline phosphatase levels; increasedblood urea nitrogen; increased percentage of granulocyte; increasedtotal white blood cell (WBC) count; increased mean absolute neutrophilcount; neutropenia; increased absolute lymphocyte count; increasedabsolute monocyte count; increased monocytes and DC in spleen (CD11b+,CD11b+c+); increased mean platelet count; increased natural killer (NK)cells in lymph node; decreased neutrophil count; decreased naturalkiller (NK) cells; decreased mean red blood cell (RBC) count, hemoglobinconcentration, and hematocrit; increased mean red cell distributionwidth; decreased mean corpuscular volume and mean corpuscularhemoglobin; decreased mean platelet count and increased platelet volume;increase B cell number in lymph node; increase in B cell subtypes inPeyer's patches; increased percentage of B cells in lymph node; increaseCD25+ cells; increased thymic DN, decreased DP T cells; increased CD19+cells in lymph node; increased CD117 in bone marrow cells; increasedmean percentage of CD4 cells; increased CD8 cells and decrease in Bcells; increased percentage CD11b+ cells in peritoneal lavage; increasedpercentage of B220+ CD11b Low CD23− cells; increased percentages ofB220− CD11 Low and CD11b− cells in peritoneal lavage; increasedpercentage of B220-CD11bHi cells in peritoneal lavage; decreasedpercentage of B220+ CD11b− CD23+ cells in peritoneal lavage; increasedpercentage of B220− CD43 Hi cells in bone marrow; increased CD11b+CD11c− cells in spleen; increase in CD62hi, CD44int subsets of CD4 andCD8 cells; increase in peritoneal CD117 cells; increase TcRbeta/CD38cells in Peyer's patches; increased percentage of TcRbeta+ cells inthymus; increased percentages of CD11b+CD11c+ in lymph node; decreasedpercentage of B220+ Hi CD23+cells in peritoneal lavage; decreasedpercentage of B220+ Med CD23−cells in peritoneal lavage; decreasedpercentages of CD62L Hi CD44 Dim CD4+ and CD8+ cells in spleen;decreased percentage of B220−CD11b Hi cells; decreased mean percentagesof CD4 and CD8 cells in lymph node and spleen; increased memory T cells[increased CD62L lo CD44hi]; decreased T cell:B cell ratio; decreasednaive T cells; decreased CD117 cells in peritoneal lavage; decreasedmean percentage of CD8 cells, increased IgG1 response to ovalbuminchallenge; increased IgG2a response to ovalbumin challenge; increasedmean serum IL-6 response to LPS challenge; increased TNF alpha responseto LPS challenge; increased serum MCP-1 response to LPS challenge;increased mean serum IgM level; increased serum IgA; increase mean serumIgG1; increased mean serum IgG3; decreased serum IgG1 response toovalbumin challenge; decreased serum IgG2a response to ovalbuminchallenge; decreased mean serum IgA level; decreased serum IgG2a level;decrease in serum IgG3 level; increased skin fibroblast proliferationrate; decreased skin fibroblast proliferation rate; increased meanpercent of total body fat and total fat mass; increased mean bodyweight; increased mean body length; increased total tissue mass (TTM);increased mean femoral midshaft cortical thickness and cross-sectionalarea; increased mean vertebral trabecular bone volume, number andconnectivity density; decreased mean percent of total body fat and totalfat mass; decreased mean body weight; decreased mean body length;decreased total tissue mass (TTM); decreased lean body mass (LBM);decreased femoral bone mineral density (BMD); decreased vertebral bonemineral density (BMD); decreased bone mineral density (BMD) in totalbody, femur and vertebrae; decreased bone mineral content (BMC);decreased bone mineral density index; decreased volumetric bone mineraldensity (vBMD); decreased mean femoral midshaft cortical thickness andcross-sectional area; decreased mean vertebral trabecular bone volume,number and connectivity density; osteopetrosis; osteoporosis; chronicinflammation in various tissues; bilateral hydronephrosis (moderate tosevere) and inflammation; “pear shaped abdomen”; bilaterally enlargedkidneys, suggesting polycystic kidney disease; degeneration of the Organof Corti; hepatocellular dysfunction; biliary obstruction;hepatosplenomegaly characterized by histiocytic infiltrate;histiocytosis in the small intestine, lymph nodes and spleen;splenomegaly, lymphadenopathy and lymphadenopathy; hyperplasia ofadenoid and tonsils; mild-moderate extra medullary hematopoiesis;homozygous mice were small, dehydrated and exhibited decreasedsubcutaneous fat depots; lipopenia; ulcerous colitis; diffuse markeddegeneration of sensory cochlear hair cells in the inner ear,characterized by a complete loss of both inner and outer cochlear haircells on the basilar membrane; gastric mucosal hyperplasia and chronicinflammation; in creased stomach weight; defective spermatogensis in thetestes; hypospermia and defective spermatozoa in the epididymus; maleinfertility; lysosomal storage disease; anemia; growth retardation;reduced viability; perinatal lethality with decreased lymphocytes andlipopenia; homozygous embryonic lethality; and heterozygous embryoniclethality.46. An agent identified by the method of Claim 26.47. The agent of Claim 46 which is an agonist or antagonist of a PRO226,PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382, PRO444,PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343, PRO1379,PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571, PRO1572,PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381, PRO4407,PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017, PRO7174,PRO9744, PRO9821, PRO9852, PRO9873, PRO10196, PRO34778, PRO20233,PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161 polypeptide.48. The agent of Claim 47, wherein the agonist is an anti-PRO226,anti-PRO257, anti-PRO268, anti-PRO290, anti-PRO36006, anti-PRO363,anti-PRO365, anti-PRO382, anti-PRO444, anti-PRO705, anti-PRO1071,anti-PRO1125, anti-PRO1134, anti-PRO1155, anti-PRO1281, anti-PRO1343,anti-PRO1379, anti-PRO1380, anti-PRO1387, anti-PRO1419, anti-PRO1433,anti-PRO1474, anti-PRO1550, anti-PRO1571, anti-PRO1572, anti-PRO1759,anti-PRO1904, anti-PRO35193, anti-PRO4341, anti-PRO4348, anti-PRO4369,anti-PRO4381, anti-PRO4407, anti-PRO4425, anti-PRO4985, anti-PRO4989,anti-PRO5737, anti-PRO5800, anti-PRO5993, anti-PRO6017, anti-PRO7174,anti-PRO9744, anti-PRO9821, anti-PRO9852, anti-PRO9873, anti-PRO10196,anti-PRO34778, anti-PRO20233, anti-PRO21956, anti-PRO57290,anti-PRO38465, anti-PRO38683 or anti-PRO85161 antibody.49. The agent of Claim 47, wherein the antagonist is an anti-PRO226,anti-PRO257, anti-PRO268, anti-PRO290, anti-PRO36006, anti-PRO363,anti-PRO365, anti-PRO382, anti-PRO444, anti-PRO705, anti-PRO1071,anti-PRO1125, anti-PRO1134, anti-PRO1155, anti-PRO1281, anti-PRO1343,anti-PRO1379, anti-PRO1380, anti-PRO1387, anti-PRO1419, anti-PRO1433,anti-PRO1474, anti-PRO1550, anti-PRO1571, anti-PRO1572, anti-PRO1759,anti-PRO1904, anti-PRO35193, anti-PRO4341, anti-PRO4348, anti-PRO4369,anti-PRO4381, anti-PRO4407, anti-PRO4425, anti-PRO4985, anti-PRO4989,anti-PRO5737, anti-PRO5800, anti-PRO5993, anti-PRO6017, anti-PRO7174,anti-PRO9744, anti-PRO9821, anti-PRO9852, anti-PRO9873, anti-PRO10196,anti-PRO34778, anti-PRO20233, anti-PRO21956, anti-PRO57290,anti-PRO38465, anti-PRO38683 or anti-PRO85161 antibody.50. A method of identifying an agent that modulates a physiologicalcharacteristic associated with a disruption of the gene which encodesfor a PRO226, PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382,PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343,PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571,PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381,PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017,PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196, PRO34778,PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161polypeptide, the method comprising:

(a) providing a non-human transgenic animal whose genome comprises adisruption of the gene which encodes for a PRO226, PRO257, PRO268,PRO290, PRO36006, PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071,PRO1125, PRO1134, PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387,PRO1419, PRO1433, PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904,PRO35193, PRO4341, PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985,PRO4989, PRO5737, PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821,PRO9852, PRO9873, PRO10196, PRO34778, PRO20233, PRO21956, PRO57290,PRO38465, PRO38683 or PRO85161 polypeptide;

(b) measuring a physiological characteristic exhibited by the non-humantransgenic animal of (a);

(c) comparing the measured physiological characteristic of (b) with thatof a gender matched wild-type animal, wherein the physiologicalcharacteristic exhibited by the non-human transgenic animal that differsfrom the physiological characteristic exhibited by the wild-type animalis identified as a physiological characteristic associated with genedisruption;

(d) administering a test agent to the non-human transgenic animal of(a); and

(e) determining whether the physiological characteristic associated withgene disruption is modulated.

51. The method of Claim 50, wherein the non-human transgenic animalexhibits at least one of the following physiological characteristicscompared with gender matched wild-type littermates: increasedanxiety-like response during open field testing; decreased anxietyduring open field testing; decreased locomotor activity during openfield testing; abnormal circadian rhythm during home-cage activitytesting (low activity during the light phase); abnormal circadian rhythmduring home-cage activity testing including decreased ambulatory counts;hypoactivity with no circadian rhythm; abnormal circadian rhythm duringhome-cage activity testing including increased ambulatory counts;increased stress induced hyperthermia; decreased stress inducedhyperthermia; impaired motor coordination during inverted screentesting; increased immobility in tail suspension (increaseddepressive-like response); increased depressive-like response duringtail suspension testing; increased immobility or decreaseddepressive-like response during tail suspension testing; decreasedstartle response during prepulse inhibition testing; no startle responseindicating deafness; or impaired hearing; decreased prepulse inhibitionwith impaired sensorimotor gating/attention; decreased responsiveness inhot plate testing; decreased latency to respond in hot plate testing;opthamological abnormalities; increased mean artery-to-vein ratio;resistance to pupil dilating drug cyclopentolate hydrochloride; squintyeyes; squint eyes with white spots; cataracts; retinal degeneration;impaired vision; decreased basal body temperature; decreased heart rate;increased mean systolic blood pressure; increased insulin sensitivity;increased mean fasting serum glucose levels; decreased mean serumglucose levels; increased mean serum cholesterol levels; decreased meanserum cholesterol levels; increased mean serum triglyceride levels;decreased mean serum triglyceride levels; enhanced glucose tolerance;impaired glucose tolerance; decreased mean serum insulin levels;increased mean serum calcium; increased urobilinogen, notable lipemia;increased albumin, alanine amino transferase, phosphorus and potassiumlevels; increased mean serum alkaline phosphatase levels; increasedblood urea nitrogen; increased percentage of granulocyte; increasedtotal white blood cell (WBC) count; increased mean absolute neutrophilcount; neutropenia; increased absolute lymphocyte count; increasedabsolute monocyte count; increased monocytes and DC in spleen (CD11b+,CD11b+c+); increased mean platelet count; increased natural killer (NK)cells in lymph node; decreased neutrophil count; decreased naturalkiller (NK) cells; decreased mean red blood cell (RBC) count, hemoglobinconcentration, and hematocrit; increased mean red cell distributionwidth; decreased mean corpuscular volume and mean corpuscularhemoglobin; decreased mean platelet count and increased platelet volume;increase B cell number in lymph node; increase in B cell subtypes inPeyer's patches; increased percentage of B cells in lymph node; increaseCD25+ cells; increased thymic DN, decreased DP T cells; increased CD19+cells in lymph node; increased CD117 in bone marrow cells; increasedmean percentage of CD4 cells; increased CD8 cells and decrease in Bcells; increased percentage CD11b+ cells in peritoneal lavage; increasedpercentage of B220+CD11b Low CD23− cells; increased percentages of B220−CD11 Low and CD11b− cells in peritoneal lavage; increased percentage ofB220-CD11bHi cells in peritoneal lavage; decreased percentage of B220+CD11b− CD23+ cells in peritoneal lavage; increased percentage of B220−CD43 Hi cells in bone marrow; increased CD11b+ CD11c− cells in spleen;increase in CD62hi, CD44int subsets of CD4 and CD8 cells; increase inperitoneal CD117 cells; increase TcRbeta/CD38 cells in Peyer's patches;increased percentage of TcRbeta+ cells in thymus; increased percentagesof CD11b+CD11c+ in lymph node; decreased percentage of B220+ Hi CD23+cells in peritoneal lavage; decreased percentage of B220+ Med CD23−cellsin peritoneal lavage; decreased percentages of CD62L Hi CD44 Dim CD4+and CD8+ cells in spleen; decreased percentage of B220−CD11b Hi cells;decreased mean percentages of CD4 and CD8 cells in lymph node andspleen; increased memory T cells [increased CD62L lo CD44hi]; decreasedT cell:B cell ratio; decreased naive T cells; decreased CD117 cells inperitoneal lavage; decreased mean percentage of CD8 cells, increasedIgG1 response to ovalbumin challenge; increased IgG2a response toovalbumin challenge; increased mean serum IL-6 response to LPSchallenge; increased TNF alpha response to LPS challenge; increasedserum MCP-1 response to LPS challenge; increased mean serum IgM level;increased serum IgA; increase mean serum IgG1; increased mean serumIgG3; decreased serum IgG1 response to ovalbumin challenge; decreasedserum IgG2a response to ovalbumin challenge; decreased mean serum IgAlevel; decreased serum IgG2a level; decrease in serum IgG3 level;increased skin fibroblast proliferation rate; decreased skin fibroblastproliferation rate; increased mean percent of total body fat and totalfat mass; increased mean body weight; increased mean body length;increased total tissue mass (TTM); increased mean femoral midshaftcortical thickness and cross-sectional area; increased mean vertebraltrabecular bone volume, number and connectivity density; decreased meanpercent of total body fat and total fat mass; decreased mean bodyweight; decreased mean body length; decreased total tissue mass (TTM);decreased lean body mass (LBM); decreased femoral bone mineral density(BMD); decreased vertebral bone mineral density (BMD); decreased bonemineral density (BMD) in total body, femur and vertebrae; decreased bonemineral content (BMC); decreased bone mineral density index; decreasedvolumetric bone mineral density (vBMD); decreased mean femoral midshaftcortical thickness and cross-sectional area; decreased mean vertebraltrabecular bone volume, number and connectivity density; osteopetrosis;osteoporosis; chronic inflammation in various tissues; bilateralhydronephrosis (moderate to severe) and inflammation; “pear shapedabdomen”; bilaterally enlarged kidneys, suggesting polycystic kidneydisease; degeneration of the Organ of Corti; hepatocellular dysfunction;biliary obstruction; hepatosplenomegaly characterized by histiocyticinfiltrate; histiocytosis in the small intestine, lymph nodes andspleen; splenomegaly, lymphadenopathy and lymphadenopathy; hyperplasiaof adenoid and tonsils; mild-moderate extra medullary hematopoiesis;homozygous mice were small, dehydrated and exhibited decreasedsubcutaneous fat depots; lipopenia; ulcerous colitis; diffuse markeddegeneration of sensory cochlear hair cells in the inner ear,characterized by a complete loss of both inner and outer cochlear haircells on the basilar membrane; gastric mucosal hyperplasia and chronicinflammation; in creased stomach weight; defective spermatogensis in thetestes; hypospermia and defective spermatozoa in the epididymus; maleinfertility; lysosomal storage disease; anemia; growth retardation;reduced viability; perinatal lethality with decreased lymphocytes andlipopenia; homozygous embryonic lethality; and heterozygous embryoniclethality.52. An agent identified by the method of Claim 50.53. The agent of Claim 52 which is an agonist or antagonist of a PRO226,PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382, PRO444,PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343, PRO1379,PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571, PRO1572,PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381, PRO4407,PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017, PRO7174,PRO9744, PRO9821, PRO9852, PRO9873, PRO10196, PRO34778, PRO20233,PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161 polypeptide.54. The agent of Claim 53, wherein the agonist is an anti-PRO226,anti-PRO257, anti-PRO268, anti-PRO290, anti-PRO36006, anti-PRO363,anti-PRO365, anti-PRO382, anti-PRO444, anti-PRO705, anti-PRO1071,anti-PRO1125, anti-PRO1134, anti-PRO1155, anti-PRO1281, anti-PRO1343,anti-PRO1379, anti-PRO1380, anti-PRO1387, anti-PRO1419, anti-PRO1433,anti-PRO1474, anti-PRO1550, anti-PRO1571, anti-PRO1572, anti-PRO1759,anti-PRO1904, anti-PRO35193, anti-PRO4341, anti-PRO4348, anti-PRO4369,anti-PRO4381, anti-PRO4407, anti-PRO4425, anti-PRO4985, anti-PRO4989,anti-PRO5737, anti-PRO5800, anti-PRO5993, anti-PRO6017, anti-PRO7174,anti-PRO9744, anti-PRO9821, anti-PRO9852, anti-PRO9873, anti-PRO10196,anti-PRO34778, anti-PRO20233, anti-PRO21956, anti-PRO57290,anti-PRO38465, anti-PRO38683 or anti-PRO85161 antibody.55. The agent of Claim 53, wherein the antagonist is an anti-PRO226,anti-PRO257, anti-PRO268, anti-PRO290, anti-PRO36006, anti-PRO363,anti-PRO365, anti-PRO382, anti-PRO444, anti-PRO705, anti-PRO1071,anti-PRO1125, anti-PRO1134, anti-PRO1155, anti-PRO1281, anti-PRO1343,anti-PRO1379, anti-PRO1380, anti-PRO1387, anti-PRO1419, anti-PRO1433,anti-PRO1474, anti-PRO1550, anti-PRO1571, anti-PRO1572, anti-PRO1759,anti-PRO1904, anti-PRO35193, anti-PRO4341, anti-PRO4348, anti-PRO4369,anti-PRO4381, anti-PRO4407, anti-PRO4425, anti-PRO4985, anti-PRO4989,anti-PRO5737, anti-PRO5800, anti-PRO5993, anti-PRO6017, anti-PRO7174,anti-PRO9744, anti-PRO9821, anti-PRO9852, anti-PRO9873, anti-PRO10196,anti-PRO34778, anti-PRO20233, anti-PRO21956, anti-PRO57290,anti-PRO38465, anti-PRO38683 or anti-PRO85161 antibody.56. A method of identifying an agent which modulates a behaviorassociated with a disruption of the gene which encodes for a PRO226,PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382, PRO444,PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343, PRO1379,PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571, PRO1572,PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381, PRO4407,PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017, PRO7174,PRO9744, PRO9821, PRO9852, PRO9873, PRO10196, PRO34778, PRO20233,PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161 polypeptide, themethod comprising:

(a) providing a non-human transgenic animal whose genome comprises adisruption of the gene which encodes for a PRO226, PRO257, PRO268,PRO290, PRO36006, PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071,PRO1125, PRO1134, PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387,PRO1419, PRO1433, PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904,PRO35193, PRO4341, PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985,PRO4989, PRO5737, PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821,PRO9852, PRO9873, PRO10196, PRO34778, PRO20233, PRO21956, PRO57290,PRO38465, PRO38683 or PRO85161 polypeptide;

(b) observing the behavior exhibited by the non-human transgenic animalof (a);

(c) comparing the observed behavior of (b) with that of a gender matchedwild-type animal, wherein the observed behavior exhibited by thenon-human transgenic animal that differs from the observed behaviorexhibited by the wild-type animal is identified as a behavior associatedwith gene disruption;

(d) administering a test agent to the non-human transgenic animal of(a); and

(e) determining whether the agent modulates the behavior associated withgene disruption.

57. The method of Claim 56, wherein the behavior is an increasedanxiety-like response during open field activity testing.

58. The method of Claim 56, wherein the behavior is a decreasedanxiety-like response during open field activity testing.

59. The method of Claim 56, wherein the behavior is an abnormalcircadian rhythm during home-cage activity testing.

60. The method of Claim 56, wherein the behavior is an enhanced motorcoordination during inverted screen testing.

61. The method of Claim 56, wherein the behavior is an impaired motorcoordination during inverted screen testing.

62. The method of Claim 56, wherein the behavior is depression,generalized anxiety disorders, attention deficit disorder, sleepdisorder, hyperactivity disorder, obsessive compulsive disorder,schizophrenia, cognitive disorders, hyperalgesia or sensory disorders.63. An agent identified by the method of Claim 56.64. The agent of Claim 63 which is an agonist or antagonist of a PRO226,PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382, PRO444,PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343, PRO1379,PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571, PRO1572,PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381, PRO4407,PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017, PRO7174,PRO9744, PRO9821, PRO9852, PRO9873, PRO10196, PRO34778, PRO20233,PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161 polypeptide.65. The agent of Claim 64, wherein the agonist is an anti-PRO226,anti-PRO257, anti-PRO268, anti-PRO290, anti-PRO36006, anti-PRO363,anti-PRO365, anti-PRO382, anti-PRO444, anti-PRO705, anti-PRO1071,anti-PRO1125, anti-PRO1134, anti-PRO1155, anti-PRO1281, anti-PRO1343,anti-PRO1379, anti-PRO1380, anti-PRO1387, anti-PRO1419, anti-PRO1433,anti-PRO1474, anti-PRO1550, anti-PRO1571, anti-PRO1572, anti-PRO1759,anti-PRO1904, anti-PRO35193, anti-PRO4341, anti-PRO4348, anti-PRO4369,anti-PRO4381, anti-PRO4407, anti-PRO4425, anti-PRO4985, anti-PRO4989,anti-PRO5737, anti-PRO5800, anti-PRO5993, anti-PRO6017, anti-PRO7174,anti-PRO9744, anti-PRO9821, anti-PRO9852, anti-PRO9873, anti-PRO10196,anti-PRO34778, anti-PRO20233, anti-PRO21956, anti-PRO57290,anti-PRO38465, anti-PRO38683 or anti-PRO85161 antibody.66. The agent of Claim 64, wherein the antagonist is an anti-PRO226,anti-PRO257, anti-PRO268, anti-PRO290, anti-PRO36006, anti-PRO363,anti-PRO365, anti-PRO382, anti-PRO444, anti-PRO705, anti-PRO1071,anti-PRO1125, anti-PRO1134, anti-PRO1155, anti-PRO1281, anti-PRO1343,anti-PRO1379, anti-PRO1380, anti-PRO1387, anti-PRO1419, anti-PRO1433,anti-PRO1474, anti-PRO1550, anti-PRO1571, anti-PRO1572, anti-PRO1759,anti-PRO1904, anti-PRO35193, anti-PRO4341, anti-PRO4348, anti-PRO4369,anti-PRO4381, anti-PRO4407, anti-PRO4425, anti-PRO4985, anti-PRO4989,anti-PRO5737, anti-PRO5800, anti-PRO5993, anti-PRO6017, anti-PRO7174,anti-PRO9744, anti-PRO9821, anti-PRO9852, anti-PRO9873, anti-PRO10196,anti-PRO34778, anti-PRO20233, anti-PRO21956, anti-PRO57290,anti-PRO38465, anti-PRO38683 or anti-PRO85161 antibody.67. A method of identifying an agent that ameliorates or modulates aneurological disorder; a cardiovascular, endothelial or angiogenicdisorder; an eye abnormality; an immunological disorder; an oncologicaldisorder; a bone metabolic abnormality or disorder; a lipid metabolicdisorder; or a developmental abnormality associated with a disruption inthe gene which encodes for a PRO226, PRO257, PRO268, PRO290, PRO36006,PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125, PRO1134,PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433,PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341,PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737,PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873,PRO10196, PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 orPRO85161 polypeptide, the method comprising:

(a) providing a non-human transgenic animal whose genome comprises adisruption of the gene which encodes for a PRO226, PRO257, PRO268,PRO290, PRO36006, PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071,PRO1125, PRO1134, PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387,PRO1419, PRO1433, PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904,PRO35193, PRO4341, PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985,PRO4989, PRO5737, PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821,PRO9852, PRO9873, PRO10196, PRO34778, PRO20233, PRO21956, PRO57290,PRO38465, PRO38683 or PRO85161 polypeptide;

(b) administering a test agent to said non-human transgenic animal; and

(c) determining whether said test agent ameliorates or modulates theneurological disorder; cardiovascular, endothelial or angiogenicdisorder; eye abnormality; immunological disorder; oncological disorder;bone metabolic abnormality or disorder; lipid metabolic disorder; ordevelopmental abnormality in the non-human transgenic animal.

68. The method of Claim 67, wherein the neurological disorder is anincreased anxiety-like response during open field activity testing.

69. The method of Claim 67, wherein the neurological disorder is adecreased anxiety-like response during open field activity testing.

70. The method of Claim 67, wherein the neurological disorder is anabnormal circadian rhythm during home-cage activity testing.

71. The method of Claim 67, wherein the neurological disorder is anenhanced motor coordination during inverted screen testing.

72. The method of Claim 67, wherein the neurological disorder is animpaired motor coordination during inverted screen testing.

73. The method of Claim 73, wherein the neurological disorder isdepression, generalized anxiety disorders, attention deficit disorder,sleep disorder, hyperactivity disorder, obsessive compulsive disorder,schizophrenia, cognitive disorders, hyperalgesia or sensory disorders.74. The method of Claim 67, wherein the eye abnormality is a retinalabnormality.75. The method of Claim 67, wherein the eye abnormality is consistentwith vision problems or blindness.76. The method of Claim 74, wherein the retinal abnormality isconsistent with retinitis pigmentosa.77. The method of Claim 74, wherein the retinal abnormality ischaracterized by retinal degeneration or retinal dysplasia.78. The method of Claim 74, wherein the retinal abnormality isconsistent with retinal dysplasia, various retinopathies, includingretinopathy of prematurity, retrolental fibroplasia, neovascularglaucoma, age-related macular degeneration, diabetic macular edema,corneal neovascularization, corneal graft neovascularization, cornealgraft rejection, retinal/choroidal neovascularization,neovascularization of the angle (rubeosis), ocular neovascular disease,vascular restenosis, arteriovenous malformations (AVM), meningioma,hemangioma, angiofibroma, thyroid hyperplasias (including Grave'sdisease), corneal and other tissue transplantation, retinal arteryobstruction or occlusion; retinal degeneration causing secondary atrophyof the retinal vasculature, retinitis pigmentosa, macular dystrophies,Stargardt's disease, congenital stationary night blindness,choroideremia, gyrate atrophy, Leber's congenital amaurosis,retinoschisis disorders, Wagner's syndrome, Usher syndromes, Zellwegersyndrome, Saldino-Mainzer syndrome, Senior-Loken syndrome, Bardet-Biedlsyndrome, Alport's syndrome, Alstrom's syndrome, Cockayne's syndrome,dysplaisa spondyloepiphysaria congentia, Flynn-Aird syndrome, Friedreichataxia, Hallgren syndrome, Marshall syndrome, Albers-Schnoberg disease,Refsum's disease, Kearns-Sayre syndrome, Waardenburg's syndrome, Alagilesyndrome, myotonic dystrophy, olivopontocerebellar atrophy, Pierre-Mariedunsdrome, Stickler syndrome, carotinemeia, cystinosis, Wolframsyndrome, Bassen-Kornzweig syndrome, abetalipoproteinemia, incontinentiapigmenti, Batten's disease, mucopolysaccharidoses, homocystinuria, ormannosidosis.79. The method of Claim 67, wherein the eye abnormality is a cataract.80. The method of Claim 79, wherein the cataract is a systemic diseasesuch as human Down's syndrome, Hallerman-Streiff syndrome, Lowesyndrome, galactosemia, Marfan syndrome, Trismoy 13-15, Alport syndrome,myotonic dystrophy, Fabry disease, hypoparathroidism or Conradisyndrome.81. The method of Claim 67, wherein the developmental abnormalitycomprises embryonic lethality or reduced viability.82. The method of Claim 67, wherein the cardiovascular, endothelial orangiogenic disorders are arterial diseases, such as diabetes mellitus;papilledema; optic atrophy; atherosclerosis; angina; myocardialinfarctions such as acute myocardial infarctions, cardiac hypertrophy,and heart failure such as congestive heart failure; hypertension;inflammatory vasculitides; Reynaud's disease and Reynaud's phenomenon;aneurysms and arterial restenosis; venous and lymphatic disorders suchas thrombophlebitis, lymphangitis, and lymphedema; peripheral vasculardisease; cancer such as vascular tumors, e.g., hemangioma (capillary andcavernous), glomus tumors, telangiectasia, bacillary angiomatosis,hemangioendothelioma, angiosarcoma, haemangiopericytoma, Kaposi'ssarcoma, lymphangioma, and lymphangiosarcoma; tumor angiogenesis; traumasuch as wounds, burns, and other injured tissue, implant fixation,scarring; ischemia reperfusion injury; rheumatoid arthritis;cerebrovascular disease; renal diseases such as acute renal failure, orosteoporosis.83. The method of Claim 67, wherein the immunological disorders aresystemic lupus erythematosis; rheumatoid arthritis; juvenile chronicarthritis; spondyloarthropathies; systemic sclerosis (scleroderma);idiopathic inflammatory myopathies (dermatomyositis, polymyositis);Sjögren's syndrome; systemic vasculitis; sarcoidosis; autoimmunehemolytic anemia (immune pancytopenia, paroxysmal nocturnalhemoglobinuria); autoimmune thrombocytopenia (idiopathicthrombocytopenic purpura, immune-mediated thrombocytopenia); thyroiditis(Grave's disease, Hashimoto's thyroiditis, juvenile lymphocyticthyroiditis, atrophic thyroiditis); diabetes mellitus; immune-mediatedrenal disease (glomerulonephritis, tubulointerstitial nephritis);demyelinating diseases of the central and peripheral nervous systemssuch as multiple sclerosis, idiopathic demyelinating polyneuropathy orGuillain-Barré syndrome, and chronic inflammatory demyelinatingpolyneuropathy; hepatobiliary diseases such as infectious hepatitis(hepatitis A, B, C, D, E and other non-hepatotropic viruses), autoimmunechronic active hepatitis, primary biliary cirrhosis, granulomatoushepatitis, and sclerosing cholangitis; inflammatory bowel disease(ulcerative colitis: Crohn's disease); gluten-sensitive enteropathy, andWhipple's disease; autoimmune or immune-mediated skin diseases includingbullous skin diseases, erythema multiforme and contact dermatitis,psoriasis; allergic diseases such as asthma, allergic rhinitis, atopicdermatitis, food hypersensitivity and urticaria; immunologic diseases ofthe lung such as eosinophilic pneumonia, idiopathic pulmonary fibrosisand hypersensitivity pneumonitis; or transplantation associated diseasesincluding graft rejection and graft-versus-host disease.84. The method of Claim 67, wherein said bone metabolic abnormality ordisorder is arthritis, osteoporosis or osteopetrosis.85. The method of Claim 67, wherein the non-human transgenic animalexhibits at least one of the following physiological characteristicscompared with gender matched wild-type littermates: increasedanxiety-like response during open field testing; decreased anxietyduring open field testing; decreased locomotor activity during openfield testing; abnormal circadian rhythm during home-cage activitytesting (low activity during the light phase); abnormal circadian rhythmduring home-cage activity testing including decreased ambulatory counts;hypoactivity with no circadian rhythm; abnormal circadian rhythm duringhome-cage activity testing including increased ambulatory counts;increased stress induced hyperthermia; decreased stress inducedhyperthermia; impaired motor coordination during inverted screentesting; increased immobility in tail suspension (increaseddepressive-like response); increased depressive-like response duringtail suspension testing; increased immobility or decreaseddepressive-like response during tail suspension testing; decreasedstartle response during prepulse inhibition testing; no startle responseindicating deafness; or impaired hearing; decreased prepulse inhibitionwith impaired sensorimotor gating/attention; decreased responsiveness inhotplate testing; decreased latency to respond in hot plate testing;opthamological abnormalities; increased mean artery-to-vein ratio;resistance to pupil dilating drug cyclopentolate hydrochloride; squintyeyes; squint eyes with white spots; cataracts; retinal degeneration;impaired vision; decreased basal body temperature; decreased heart rate;increased mean systolic blood pressure; increased insulin sensitivity;increased mean fasting serum glucose levels; decreased mean serumglucose levels; increased mean serum cholesterol levels; decreased meanserum cholesterol levels; increased mean serum triglyceride levels;decreased mean serum triglyceride levels; enhanced glucose tolerance;impaired glucose tolerance; decreased mean serum insulin levels;increased mean serum calcium; increased urobilinogen, notable lipemia;increased albumin, alanine amino transferase, phosphorus and potassiumlevels; increased mean serum alkaline phosphatase levels; increasedblood urea nitrogen; increased percentage of granulocyte; increasedtotal white blood cell (WBC) count; increased mean absolute neutrophilcount; neutropenia; increased absolute lymphocyte count; increasedabsolute monocyte count; increased monocytes and DC in spleen (CD11b+,CD11b+c+); increased mean platelet count; increased natural killer (NK)cells in lymph node; decreased neutrophil count; decreased naturalkiller (NK) cells; decreased mean red blood cell (RBC) count, hemoglobinconcentration, and hematocrit; increased mean red cell distributionwidth; decreased mean corpuscular volume and mean corpuscularhemoglobin; decreased mean platelet count and increased platelet volume;increase B cell number in lymph node; increase in B cell subtypes inPeyer's patches; increased percentage of B cells in lymph node; increaseCD25+ cells; increased thymic DN, decreased DP T cells; increased CD19+cells in lymph node; increased CD117 in bone marrow cells; increasedmean percentage of CD4 cells; increased CD8 cells and decrease in Bcells; increased percentage CD11b+ cells in peritoneal lavage; increasedpercentage of B220+CD11b Low CD23− cells; increased percentages of B220−CD11 Low and CD11b− cells in peritoneal lavage; increased percentage ofB220-CD11bHi cells in peritoneal lavage; decreased percentage of B220+CD11b− CD23+ cells in peritoneal lavage; increased percentage of B220−CD43 Hi cells in bone marrow; increased CD11b+CD11c− cells in spleen;increase in CD62hi, CD44int subsets of CD4 and CD8 cells; increase inperitoneal CD117 cells; increase TcRbeta/CD38 cells in Peyer's patches;increased percentage of TcRbeta+ cells in thymus; increased percentagesof CD11b+CD11c+ in lymph node; decreased percentage of B220+ Hi CD23+cells in peritoneal lavage; decreased percentage of B220+ Med CD23−cellsin peritoneal lavage; decreased percentages of CD62L Hi CD44 Dim CD4+and CD8+ cells in spleen; decreased percentage of B220−CD11b Hi cells;decreased mean percentages of CD4 and CD8 cells in lymph node andspleen; increased memory T cells [increased CD62L lo CD44hi]; decreasedT cell:B cell ratio; decreased naive T cells; decreased CD117 cells inperitoneal lavage; decreased mean percentage of CD8 cells, increasedIgG1 response to ovalbumin challenge; increased IgG2a response toovalbumin challenge; increased mean serum IL-6 response to LPSchallenge; increased TNF alpha response to LPS challenge; increasedserum MCP-1 response to LPS challenge; increased mean serum IgM level;increased serum IgA; increase mean serum IgG1; increased mean serumIgG3; decreased serum IgG1 response to ovalbumin challenge; decreasedserum IgG2a response to ovalbumin challenge; decreased mean serum IgAlevel; decreased serum IgG2a level; decrease in serum IgG3 level;increased skin fibroblast proliferation rate; decreased skin fibroblastproliferation rate; increased mean percent of total body fat and totalfat mass; increased mean body weight; increased mean body length;increased total tissue mass (TTM); increased mean femoral midshaftcortical thickness and cross-sectional area; increased mean vertebraltrabecular bone volume, number and connectivity density; decreased meanpercent of total body fat and total fat mass; decreased mean bodyweight; decreased mean body length; decreased total tissue mass (TTM);decreased lean body mass (LBM); decreased femoral bone mineral density(BMD); decreased vertebral bone mineral density (BMD); decreased bonemineral density (BMD) in total body, femur and vertebrae; decreased bonemineral content (BMC); decreased bone mineral density index; decreasedvolumetric bone mineral density (vBMD); decreased mean femoral midshaftcortical thickness and cross-sectional area; decreased mean vertebraltrabecular bone volume, number and connectivity density; osteopetrosis;osteoporosis; chronic inflammation in various tissues; bilateralhydronephrosis (moderate to severe) and inflammation; “pear shapedabdomen”; bilaterally enlarged kidneys, suggesting polycystic kidneydisease; degeneration of the Organ of Corti; hepatocellular dysfunction;biliary obstruction; hepatosplenomegaly characterized by histiocyticinfiltrate; histiocytosis in the small intestine, lymph nodes andspleen; splenomegaly, lymphadenopathy and lymphadenopathy; hyperplasiaof adenoid and tonsils; mild-moderate extra medullary hematopoiesis;homozygous mice were small, dehydrated and exhibited decreasedsubcutaneous fat depots; lipopenia; ulcerous colitis; diffuse markeddegeneration of sensory cochlear hair cells in the inner ear,characterized by a complete loss of both inner and outer cochlear haircells on the basilar membrane; gastric mucosal hyperplasia and chronicinflammation; in creased stomach weight; defective spermatogensis in thetestes; hypospermia and defective spermatozoa in the epididymus; maleinfertility; lysosomal storage disease; anemia; growth retardation;reduced viability; perinatal lethality with decreased lymphocytes andlipopenia; homozygous embryonic lethality; and heterozygous embryoniclethality.86. An agent identified by the method of Claim 67.87. The agent of Claim 86 which is an agonist or antagonist of a PRO226,PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382, PRO444,PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343, PRO1379,PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571, PRO1572,PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381, PRO4407,PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017, PRO7174,PRO9744, PRO9821, PRO9852, PRO9873, PRO10196, PRO34778, PRO20233,PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161 polypeptide.88. The agent of Claim 87, wherein the agonist is an anti-PRO226,anti-PRO257, anti-PRO268, anti-PRO290, anti-PRO36006, anti-PRO363,anti-PRO365, anti-PRO382, anti-PRO444, anti-PRO705, anti-PRO1071,anti-PRO1125, anti-PRO1134, anti-PRO1155, anti-PRO1281, anti-PRO1343,anti-PRO1379, anti-PRO1380, anti-PRO1387, anti-PRO1419, anti-PRO1433,anti-PRO1474, anti-PRO1550, anti-PRO1571, anti-PRO1572, anti-PRO1759,anti-PRO1904, anti-PRO35193, anti-PRO4341, anti-PRO4348, anti-PRO4369,anti-PRO4381, anti-PRO4407, anti-PRO4425, anti-PRO4985, anti-PRO4989,anti-PRO5737, anti-PRO5800, anti-PRO5993, anti-PRO6017, anti-PRO7174,anti-PRO9744, anti-PRO9821, anti-PRO9852, anti-PRO9873, anti-PRO10196,anti-PRO34778, anti-PRO20233, anti-PRO21956, anti-PRO57290,anti-PRO38465, anti-PRO38683 or anti-PRO85161 antibody.89. The agent of Claim 87, wherein the antagonist is an anti-PRO226,anti-PRO257, anti-PRO268, anti-PRO290, anti-PRO36006, anti-PRO363,anti-PRO365, anti-PRO382, anti-PRO444, anti-PRO705, anti-PRO1071,anti-PRO1125, anti-PRO134, anti-PRO1155, anti-PRO1281, anti-PRO1343,anti-PRO1379, anti-PRO1380, anti-PRO1387, anti-PRO1419, anti-PRO1433,anti-PRO1474, anti-PRO1550, anti-PRO1571, anti-PRO1572, anti-PRO1759,anti-PRO1904, anti-PRO35193, anti-PRO4341, anti-PRO4348, anti-PRO4369,anti-PRO4381, anti-PRO4407, anti-PRO4425, anti-PRO4985, anti-PRO4989,anti-PRO5737, anti-PRO5800, anti-PRO5993, anti-PRO6017, anti-PRO7174,anti-PRO9744, anti-PRO9821, anti-PRO9852, anti-PRO9873, anti-PRO10196,anti-PRO34778, anti-PRO20233, anti-PRO21956, anti-PRO57290,anti-PRO38465, anti-PRO38683 or anti-PRO85161 antibody.90. A therapeutic agent identified by the method of Claim 67.91. A method of identifying an agent that modulates the expression of aPRO226, PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382,PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343,PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571,PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381,PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017,PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196, PRO34778,PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161polypeptide, the method comprising:

(a) contacting a test agent with a host cell expressing a PRO226,PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382, PRO444,PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343, PRO1379,PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571, PRO1572,PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381, PRO4407,PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017, PRO7174,PRO9744, PRO9821, PRO9852, PRO9873, PRO10196, PRO34778, PRO20233,PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161 polypeptide; and

(b) determining whether the test agent modulates the expression of thePRO226, PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382,PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343,PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571,PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381,PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017,PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196, PRO34778,PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161 polypeptideby the host cell.

92. An agent identified by the method of Claim 91.

93. The agent of Claim 92 which is an agonist or antagonist of a PRO226,PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382, PRO444,PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343, PRO1379,PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571, PRO1572,PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381, PRO4407,PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017, PRO7174,PRO9744, PRO9821, PRO9852, PRO9873, PRO10196, PRO34778, PRO20233,PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161 polypeptide.94. The agent of Claim 93, wherein the agonist is an anti-PRO226,anti-PRO257, anti-PRO268, anti-PRO290, anti-PRO36006, anti-PRO363,anti-PRO365, anti-PRO382, anti-PRO444, anti-PRO705, anti-PRO1071,anti-PRO1125, anti-PRO1134, anti-PRO1155, anti-PRO1281, anti-PRO1343,anti-PRO1379, anti-PRO1380, anti-PRO1387, anti-PRO1419, anti-PRO1433,anti-PRO1474, anti-PRO1550, anti-PRO1571, anti-PRO1572, anti-PRO1759,anti-PRO1904, anti-PRO35193, anti-PRO4341, anti-PRO4348, anti-PRO4369,anti-PRO4381, anti-PRO4407, anti-PRO4425, anti-PRO4985, anti-PRO4989,anti-PRO5737, anti-PRO5800, anti-PRO5993, anti-PRO6017, anti-PRO7174,anti-PRO9744, anti-PRO9821, anti-PRO9852, anti-PRO9873, anti-PRO10196,anti-PRO34778, anti-PRO20233, anti-PRO21956, anti-PRO57290,anti-PRO38465, anti-PRO38683 or anti-PRO85161 antibody.95. The agent of Claim 93, wherein the antagonist is an anti-PRO226,anti-PRO257, anti-PRO268, anti-PRO290, anti-PRO36006, anti-PRO363,anti-PRO365, anti-PRO382, anti-PRO444, anti-PRO705, anti-PRO1071,anti-PRO1125, anti-PRO134, anti-PRO1155, anti-PRO1281, anti-PRO1343,anti-PRO1379, anti-PRO1380, anti-PRO1387, anti-PRO1419, anti-PRO1433,anti-PRO1474, anti-PRO1550, anti-PRO1571, anti-PRO1572, anti-PRO1759,anti-PRO1904, anti-PRO35193, anti-PRO4341, anti-PRO4348, anti-PRO4369,anti-PRO4381, anti-PRO4407, anti-PRO4425, anti-PRO4985, anti-PRO4989,anti-PRO5737, anti-PRO5800, anti-PRO5993, anti-PRO6017, anti-PRO7174,anti-PRO9744, anti-PRO9821, anti-PRO9852, anti-PRO9873, anti-PRO10196,anti-PRO34778, anti-PRO20233, anti-PRO21956, anti-PRO57290,anti-PRO38465, anti-PRO38683 or anti-PRO85161 antibody.96. A method of evaluating a therapeutic agent capable of affecting acondition associated with a disruption of a gene which encodes for aPRO226, PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382,PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343,PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571,PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381,PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017,PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196, PRO34778,PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161polypeptide, the method comprising:

(a) providing a non-human transgenic animal whose genome comprises adisruption of the gene which encodes for the PRO226, PRO257, PRO268,PRO290, PRO36006, PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071,PRO1125, PRO1134, PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387,PRO1419, PRO1433, PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904,PRO35193, PRO4341, PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985,PRO4989, PRO5737, PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821,PRO9852, PRO9873, PRO10196, PRO34778, PRO20233, PRO21956, PRO57290,PRO38465, PRO38683 or PRO85161 polypeptide;

(b) measuring a physiological characteristic of the non-human transgenicanimal of (a);

(c) comparing the measured physiological characteristic of (b) with thatof a gender matched wild-type animal, wherein the physiologicalcharacteristic of the non-human transgenic animal that differs from thephysiological characteristic of the wild-type animal is identified as acondition resulting from the gene disruption in the non-human transgenicanimal;

(d) administering a test agent to the non-human transgenic animal of(a); and

(e) evaluating the effects of the test agent on the identified conditionassociated with gene disruption in the non-human transgenic animal.

97. The method of Claim 96, wherein the condition is a neurologicaldisorder; a cardiovascular, endothelial or angiogenic disorder; an eyeabnormality; an immunological disorder; an oncological disorder; a bonemetabolic abnormality or disorder; a lipid metabolic disorder; or adevelopmental abnormality.98. A therapeutic agent identified by the method of Claim 96.99. The therapeutic agent of Claim 98 which is an agonist or antagonistof a PRO226, PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382,PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343,PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571,PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381,PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017,PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196, PRO34778,PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161polypeptide.100. The therapeutic agent of Claim 99, wherein the agonist is ananti-PRO226, anti-PRO257, anti-PRO268, anti-PRO290, anti-PRO36006,anti-PRO363, anti-PRO365, anti-PRO382, anti-PRO444, anti-PRO705,anti-PRO1071, anti-PRO1125, anti-PRO1134, anti-PRO1155, anti-PRO1281,anti-PRO1343, anti-PRO1379, anti-PRO1380, anti-PRO1387, anti-PRO1419,anti-PRO1433, anti-PRO1474, anti-PRO1550, anti-PRO1571, anti-PRO1572,anti-PRO1759, anti-PRO1904, anti-PRO35193, anti-PRO4341, anti-PRO4348,anti-PRO4369, anti-PRO4381, anti-PRO4407, anti-PRO4425, anti-PRO4985,anti-PRO4989, anti-PRO5737, anti-PRO5800, anti-PRO5993, anti-PRO6017,anti-PRO7174, anti-PRO9744, anti-PRO9821, anti-PRO9852, anti-PRO9873,anti-PRO10196, anti-PRO34778, anti-PRO20233, anti-PRO21956,anti-PRO57290, anti-PRO38465, anti-PRO38683 or anti-PRO85161 antibody.101. The therapeutic agent of Claim 99, wherein the antagonist is ananti-PRO226, anti-PRO257, anti-PRO268, anti-PRO290, anti-PRO36006,anti-PRO363, anti-PRO365, anti-PRO382, anti-PRO444, anti-PRO705,anti-PRO1071, anti-PRO1125, anti-PRO1134, anti-PRO1155, anti-PRO1281,anti-PRO1343, anti-PRO1379, anti-PRO1380, anti-PRO1387, anti-PRO1419,anti-PRO1433, anti-PRO1474, anti-PRO1550, anti-PRO1571, anti-PRO1572,anti-PRO1759, anti-PRO1904, anti-PRO35193, anti-PRO4341, anti-PRO4348,anti-PRO4369, anti-PRO4381, anti-PRO4407, anti-PRO4425, anti-PRO4985,anti-PRO4989, anti-PRO5737, anti-PRO5800, anti-PRO5993, anti-PRO6017,anti-PRO7174, anti-PRO9744, anti-PRO9821, anti-PRO9852, anti-PRO9873,anti-PRO10196, anti-PRO34778, anti-PRO20233, anti-PRO21956,anti-PRO57290, anti-PRO38465, anti-PRO38683 or anti-PRO85161 antibody.102. A pharmaceutical composition comprising the therapeutic agent ofClaim 98.103. A method of treating or preventing or ameliorating a neurologicaldisorder; cardiovascular, endothelial or angiogenic disorder;immunological disorder; oncological disorder; bone metabolic abnormalityor disorder, or embryonic lethality associated with the disruption of agene which encodes for a PRO226, PRO257, PRO268, PRO290, PRO36006,PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125, PRO1134,PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433,PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341,PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737,PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873,PRO10196, PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 orPRO85161 polypeptide, the method comprising administering to a subjectin need of such treatment whom may already have the disorder, or may beprone to have the disorder or may be in whom the disorder is to beprevented, a therapeutically effective amount of the therapeutic agentof Claim 94, or agonists or antagonists thereof, thereby effectivelytreating or preventing or ameliorating said disorder.104. The method of Claim 103, wherein the neurological disorder is anincreased anxiety-like response during open field activity testing.105. The method of Claim 103, wherein the neurological disorder is adecreased anxiety-like response during open field activity testing.106. The method of Claim 103, wherein the neurological disorder is anabnormal circadian rhythm during home-cage activity testing.107. The method of Claim 103, wherein the neurological disorder is anenhanced motor coordination during inverted screen testing.108. The method of Claim 103, wherein the neurological disorder is animpaired motor coordination during inverted screen testing.109. The method of Claim 103, wherein the neurological disorder isdepression, generalized anxiety disorders, attention deficit disorder,sleep disorder, hyperactivity disorder, obsessive compulsive disorder,schizophrenia, cognitive disorders, hyperalgesia or sensory disorders.110. The method of Claim 103, wherein the eye abnormality is a retinalabnormality.111. The method of Claim 103, wherein the eye abnormality is consistentwith vision problems or blindness.112. The method of Claim 110, wherein the retinal abnormality isconsistent with retinitis pigmentosa.113. The method of Claim 110, wherein the retinal abnormality ischaracterized by retinal degeneration or retinal dysplasia.114. The method of Claim 110, wherein the retinal abnormality isconsistent with retinal dysplasia, various retinopathies, includingretinopathy of prematurity, retrolental fibroplasia, neovascularglaucoma, age-related macular degeneration, diabetic macular edema,corneal neovascularization, corneal graft neovascularization, cornealgraft rejection, retinal/choroidal neovascularization,neovascularization of the angle (rubeosis), ocular neovascular disease,vascular restenosis, arteriovenous malformations (AVM), meningioma,hemangioma, angiofibroma, thyroid hyperplasias (including Grave'sdisease), corneal and other tissue transplantation, retinal arteryobstruction or occlusion; retinal degeneration causing secondary atrophyof the retinal vasculature, retinitis pigmentosa, macular dystrophies,Stargardt's disease, congenital stationary night blindness,choroideremia, gyrate atrophy, Leber's congenital amaurosis,retinoschisis disorders, Wagner's syndrome, Usher syndromes, Zellwegersyndrome, Saldino-Mainzer syndrome, Senior-Loken syndrome, Bardet-Biedlsyndrome, Alport's syndrome, Alstrom's syndrome, Cockayne's syndrome,dysplaisa spondyloepiphysaria congentia, Flynn-Aird syndrome, Friedreichataxia, Hallgren syndrome, Marshall syndrome, Albers-Schnoberg disease,Refsum's disease, Kearns-Sayre syndrome, Waardenburg's syndrome, Alagilesyndrome, myotonic dystrophy, olivopontocerebellar atrophy, Pierre-Mariedunsdrome, Stickler syndrome, carotinemeia, cystinosis, Wolframsyndrome, Bassen-Kornzweig syndrome, abetalipoproteinemia, incontinentiapigmenti, Batten's disease, mucopolysaccharidoses, homocystinuria, ormannosidosis.115. The method of Claim 103, wherein the eye abnormality is a cataract.116. The method of Claim 115, wherein the cataract is a systemic diseasesuch as human Down's syndrome, Hallerman-Streiff syndrome, Lowesyndrome, galactosemia, Marfan syndrome, Trismoy 13-15, Alport syndrome,myotonic dystrophy, Fabry disease, hypoparathroidism or Conradisyndrome.117. The method of Claim 103, wherein the developmental abnormalitycomprises embryonic lethality or reduced viability.118. The method of Claim 103, wherein the cardiovascular, endothelial orangiogenic disorders are arterial diseases, such as diabetes mellitus;papilledema; optic atrophy; atherosclerosis; angina; myocardialinfarctions such as acute myocardial infarctions, cardiac hypertrophy,and heart failure such as congestive heart failure; hypertension;inflammatory vasculitides; Reynaud's disease and Reynaud's phenomenon;aneurysms and arterial restenosis; venous and lymphatic disorders suchas thrombophlebitis, lymphangitis, and lymphedema; peripheral vasculardisease; cancer such as vascular tumors, e.g., hemangioma (capillary andcavernous), glomus tumors, telangiectasia, bacillary angiomatosis,hemangioendothelioma, angiosarcoma, haemangiopericytoma, Kaposi'ssarcoma, lymphangioma, and lymphangiosarcoma; tumor angiogenesis; traumasuch as wounds, burns, and other injured tissue, implant fixation,scarring; ischemia reperfusion injury; rheumatoid arthritis;cerebrovascular disease; renal diseases such as acute renal failure, orosteoporosis.119. The method of Claim 103, wherein the immunological disorders aresystemic lupus erythematosis; rheumatoid arthritis; juvenile chronicarthritis; spondyloarthropathies; systemic sclerosis (scleroderma);idiopathic inflammatory myopathies (dermatomyositis, polymyositis);Sjögren's syndrome; systemic vasculitis; sarcoidosis; autoimmunehemolytic anemia (immune pancytopenia, paroxysmal nocturnalhemoglobinuria); autoimmune thrombocytopenia (idiopathicthrombocytopenic purpura, immune-mediated thrombocytopenia); thyroiditis(Grave's disease, Hashimoto's thyroiditis, juvenile lymphocyticthyroiditis, atrophic thyroiditis); diabetes mellitus; immune-mediatedrenal disease (glomerulonephritis, tubulointerstitial nephritis);demyelinating diseases of the central and peripheral nervous systemssuch as multiple sclerosis, idiopathic demyelinating polyneuropathy orGuillain-Barré syndrome, and chronic inflammatory demyelinatingpolyneuropathy; hepatobiliary diseases such as infectious hepatitis(hepatitis A, B, C, D, E and other non-hepatotropic viruses), autoimmunechronic active hepatitis, primary biliary cirrhosis, granulomatoushepatitis, and sclerosing cholangitis; inflammatory bowel disease(ulcerative colitis: Crohn's disease); gluten-sensitive enteropathy, andWhipple's disease; autoimmune or immune-mediated skin diseases includingbullous skin diseases, erythema multiforme and contact dermatitis,psoriasis; allergic diseases such as asthma, allergic rhinitis, atopicdermatitis, food hypersensitivity and urticaria; immunologic diseases ofthe lung such as eosinophilic pneumonia, idiopathic pulmonary fibrosisand hypersensitivity pneumonitis; or transplantation associated diseasesincluding graft rejection and graft-versus-host disease.120. The method of Claim 103, wherein said bone metabolic abnormality ordisorder is arthritis, osteoporosis or osteopetrosis.121. A method of identifying an agent that ameliorates or modulates aneurological disorder; a cardiovascular, endothelial or angiogenicdisorder; an eye abnormality; an immunological disorder; an oncologicaldisorder; a bone metabolic abnormality or disorder; a lipid metabolicdisorder; or a developmental abnormality associated with a disruption inthe gene which encodes for a PRO226, PRO257, PRO268, PRO290, PRO36006,PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125, PRO1134,PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433,PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341,PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737,PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873,PRO10196, PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 orPRO85161 polypeptide, the method comprising:

(a) providing a non-human transgenic animal cell culture, each cell ofsaid culture comprising a disruption of the gene which encodes for aPRO226, PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382,PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343,PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571,PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381,PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017,PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196, PRO34778,PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161polypeptide;

(b) administering a test agent to said cell culture; and

(c) determining whether said test agent ameliorates or modulates theneurological disorder; cardiovascular, endothelial or angiogenicdisorder; eye abnormality; immunological disorder; oncological disorder;bone metabolic abnormality or disorder; lipid metabolic disorder; ordevelopmental abnormality in said cell culture.

122. The method of Claim 121, wherein the neurological disorder is anincreased anxiety-like response during open field activity testing.

123. The method of Claim 121, wherein the neurological disorder is adecreased anxiety-like response during open field activity testing.

124. The method of Claim 121, wherein the neurological disorder is anabnormal circadian rhythm during home-cage activity testing.

125. The method of Claim 121, wherein the neurological disorder is anenhanced motor coordination during inverted screen testing.

126. The method of Claim 121, wherein the neurological disorder is animpaired motor coordination during inverted screen testing.

127. The method of Claim 121, wherein the neurological disorder isdepression, generalized anxiety disorders, attention deficit disorder,sleep disorder, hyperactivity disorder, obsessive compulsive disorder,schizophrenia, cognitive disorders, hyperalgesia or sensory disorders.128. The method of Claim 121, wherein the eye abnormality is a retinalabnormality.129. The method of Claim 121, wherein the eye abnormality is consistentwith vision problems or blindness.130. The method of Claim 128, wherein the retinal abnormality isconsistent with retinitis pigmentosa.131. The method of Claim 128, wherein the retinal abnormality ischaracterized by retinal degeneration or retinal dysplasia.132. The method of Claim 128, wherein the retinal abnormality isconsistent with retinal dysplasia, various retinopathies, includingretinopathy of prematurity, retrolental fibroplasia, neovascularglaucoma, age-related macular degeneration, diabetic macular edema,corneal neovascularization, corneal graft neovascularization, cornealgraft rejection, retinal/choroidal neovascularization,neovascularization of the angle (rubeosis), ocular neovascular disease,vascular restenosis, arteriovenous malformations (AVM), meningioma,hemangioma, angiofibroma, thyroid hyperplasias (including Grave'sdisease), corneal and other tissue transplantation, retinal arteryobstruction or occlusion; retinal degeneration causing secondary atrophyof the retinal vasculature, retinitis pigmentosa, macular dystrophies,Stargardt's disease, congenital stationary night blindness,choroideremia, gyrate atrophy, Leber's congenital amaurosis,retinoschisis disorders, Wagner's syndrome, Usher syndromes, Zellwegersyndrome, Saldino-Mainzer syndrome, Senior-Loken syndrome, Bardet-Biedlsyndrome, Alport's syndrome, Alstrom's syndrome, Cockayne's syndrome,dysplaisa spondyloepiphysaria congentia, Flynn-Aird syndrome, Friedreichataxia, Hallgren syndrome, Marshall syndrome, Albers-Schnoberg disease,Refsum's disease, Kearns-Sayre syndrome, Waardenburg's syndrome, Alagilesyndrome, myotonic dystrophy, olivopontocerebellar atrophy, Pierre-Mariedunsdrome, Stickler syndrome, carotinemeia, cystinosis, Wolframsyndrome, Bassen-Kornzweig syndrome, abetalipoproteinemia, incontinentiapigmenti, Batten's disease, mucopolysaccharidoses, homocystinuria, ormannosidosis.133. The method of Claim 121, wherein the eye abnormality is a cataract.134. The method of Claim 133, wherein the cataract is a systemic diseasesuch as human Down's syndrome, Hallerman-Streiff syndrome, Lowesyndrome, galactosemia, Marfan syndrome, Trismoy 13-15, Alport syndrome,myotonic dystrophy, Fabry disease, hypoparathroidism or Conradisyndrome.135. The method of Claim 121, wherein the developmental abnormalitycomprises embryonic lethality or reduced viability.136. The method of Claim 121, wherein the cardiovascular, endothelial orangiogenic disorders are arterial diseases, such as diabetes mellitus;papilledema; optic atrophy; atherosclerosis; angina; myocardialinfarctions such as acute myocardial infarctions, cardiac hypertrophy,and heart failure such as congestive heart failure; hypertension;inflammatory vasculitides; Reynaud's disease and Reynaud's phenomenon;aneurysms and arterial restenosis; venous and lymphatic disorders suchas thrombophlebitis, lymphangitis, and lymphedema; peripheral vasculardisease; cancer such as vascular tumors, e.g., hemangioma (capillary andcavernous), glomus tumors, telangiectasia, bacillary angiomatosis,hemangioendothelioma, angiosarcoma, haemangiopericytoma, Kaposi'ssarcoma, lymphangioma, and lymphangiosarcoma; tumor angiogenesis; traumasuch as wounds, burns, and other injured tissue, implant fixation,scarring; ischemia reperfusion injury; rheumatoid arthritis;cerebrovascular disease; renal diseases such as acute renal failure, orosteoporosis.137. The method of Claim 121, wherein the immunological disorders aresystemic lupus erythematosis; rheumatoid arthritis; juvenile chronicarthritis; spondyloarthropathies; systemic sclerosis (scleroderma);idiopathic inflammatory myopathies (dermatomyositis, polymyositis);Sjögren's syndrome; systemic vasculitis; sarcoidosis; autoimmunehemolytic anemia (immune pancytopenia, paroxysmal nocturnalhemoglobinuria); autoimmune thrombocytopenia (idiopathicthrombocytopenic purpura, immune-mediated thrombocytopenia); thyroiditis(Grave's disease, Hashimoto's thyroiditis, juvenile lymphocyticthyroiditis, atrophic thyroiditis); diabetes mellitus; immune-mediatedrenal disease (glomerulonephritis, tubulointerstitial nephritis);demyelinating diseases of the central and peripheral nervous systemssuch as multiple sclerosis, idiopathic demyelinating polyneuropathy orGuillain-Barré syndrome, and chronic inflammatory demyelinatingpolyneuropathy; hepatobiliary diseases such as infectious hepatitis(hepatitis A, B, C, D, E and other non-hepatotropic viruses), autoimmunechronic active hepatitis, primary biliary cirrhosis, granulomatoushepatitis, and sclerosing cholangitis; inflammatory bowel disease(ulcerative colitis: Crohn's disease); gluten-sensitive enteropathy, andWhipple's disease; autoimmune or immune-mediated skin diseases includingbullous skin diseases, erythema multiforme and contact dermatitis,psoriasis; allergic diseases such as asthma, allergic rhinitis, atopicdermatitis, food hypersensitivity and urticaria; immunologic diseases ofthe lung such as eosinophilic pneumonia, idiopathic pulmonary fibrosisand hypersensitivity pneumonitis; or transplantation associated diseasesincluding graft rejection and graft-versus-host disease.138. The method of Claim 121, wherein said bone metabolic abnormality ordisorder is arthritis, osteoporosis or osteopetrosis.139. An agent identified by the method of Claim 121.140. The agent of Claim 139 which is an agonist or antagonist of aPRO226, PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382,PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343,PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571,PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381,PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017,PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196, PRO34778,PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161polypeptide.141. The agent of Claim 140, wherein the agonist is an anti-PRO226,anti-PRO257, anti-PRO268, anti-PRO290, anti-PRO36006, anti-PRO363,anti-PRO365, anti-PRO382, anti-PRO444, anti-PRO705, anti-PRO1071,anti-PRO1125, anti-PRO1134, anti-PRO1155, anti-PRO1281, anti-PRO1343,anti-PRO1379, anti-PRO1380, anti-PRO1387, anti-PRO1419, anti-PRO1433,anti-PRO1474, anti-PRO1550, anti-PRO1571, anti-PRO1572, anti-PRO1759,anti-PRO1904, anti-PRO35193, anti-PRO4341, anti-PRO4348, anti-PRO4369,anti-PRO4381, anti-PRO4407, anti-PRO4425, anti-PRO4985, anti-PRO4989,anti-PRO5737, anti-PRO5800, anti-PRO5993, anti-PRO6017, anti-PRO7174,anti-PRO9744, anti-PRO9821, anti-PRO9852, anti-PRO9873, anti-PRO10196,anti-PRO34778, anti-PRO20233, anti-PRO21956, anti-PRO57290,anti-PRO38465, anti-PRO38683 or anti-PRO85161 antibody.142. The agent of Claim 140, wherein the antagonist is an anti-PRO226,anti-PRO257, anti-PRO268, anti-PRO290, anti-PRO36006, anti-PRO363,anti-PRO365, anti-PRO382, anti-PRO444, anti-PRO705, anti-PRO1071,anti-PRO1125, anti-PRO1134, anti-PRO1155, anti-PRO1281, anti-PRO1343,anti-PRO1379, anti-PRO1380, anti-PRO1387, anti-PRO1419, anti-PRO1433,anti-PRO1474, anti-PRO1550, anti-PRO1571, anti-PRO1572, anti-PRO1759,anti-PRO1904, anti-PRO35193, anti-PRO4341, anti-PRO4348, anti-PRO4369,anti-PRO4381, anti-PRO4407, anti-PRO4425, anti-PRO4985, anti-PRO4989,anti-PRO5737, anti-PRO5800, anti-PRO5993, anti-PRO6017, anti-PRO7174,anti-PRO9744, anti-PRO9821, anti-PRO9852, anti-PRO9873, anti-PRO10196,anti-PRO34778, anti-PRO20233, anti-PRO21956, anti-PRO57290,anti-PRO38465, anti-PRO38683 or anti-PRO85161 antibody.143. A therapeutic agent identified by the method of Claim 121.144. A method of modulating a phenotype associated with a disruption ofa gene which encodes for a PRO226, PRO257, PRO268, PRO290, PRO36006,PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125, PRO1134,PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433,PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341,PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737,PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873,PRO10196, PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 orPRO85161 polypeptide, the method comprising administering to a subjectwhom may already have the phenotype, or may be prone to have thephenotype or may be in whom the phenotype is to be prevented, aneffective amount of the agent of Claim 46, or agonists or antagoniststhereof, thereby effectively modulating the phenotype.145. A method of modulating a physiological characteristic associatedwith a disruption of a gene which encodes for a PRO226, PRO257, PRO268,PRO290, PRO36006, PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071,PRO1125, PRO1134, PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387,PRO1419, PRO1433, PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904,PRO35193, PRO4341, PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985,PRO4989, PRO5737, PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821,PRO9852, PRO9873, PRO10196, PRO34778, PRO20233, PRO21956, PRO57290,PRO38465, PRO38683 or PRO85161 polypeptide, the method comprisingadministering to a subject whom may already exhibit the physiologicalcharacteristic, or may be prone to exhibit the physiologicalcharacteristic or may be in whom the physiological characteristic is tobe prevented, an effective amount of the agent of Claim 52, or agonistsor antagonists thereof, thereby effectively modulating the physiologicalcharacteristic.146. A method of modulating a behavior associated with a disruption of agene which encodes for a PRO226, PRO257, PRO268, PRO290, PRO36006,PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125, PRO1134,PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433,PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341,PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737,PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873,PRO10196, PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 orPRO85161 polypeptide, the method comprising administering to a subjectwhom may already exhibit the behavior, or may be prone to exhibit thebehavior or may be in whom the exhibited behavior is to be prevented, aneffective amount of the agent of Claim 63, or agonists or antagoniststhereof, thereby effectively modulating the behavior.147. A method of modulating the expression of a PRO226, PRO257, PRO268,PRO290, PRO36006, PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071,PRO1125, PRO1134, PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387,PRO1419, PRO1433, PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904,PRO35193, PRO4341, PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985,PRO4989, PRO5737, PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821,PRO9852, PRO9873, PRO10196, PRO34778, PRO20233, PRO21956, PRO57290,PRO38465, PRO38683 or PRO85161 polypeptide, the method comprisingadministering to a host cell expressing said PRO226, PRO257, PRO268,PRO290, PRO36006, PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071,PRO1125, PRO1134, PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387,PRO1419, PRO1433, PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904,PRO35193, PRO4341, PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985,PRO4989, PRO5737, PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821,PRO9852, PRO9873, PRO10196, PRO34778, PRO20233, PRO21956, PRO57290,PRO38465, PRO38683 or PRO85161 polypeptide, an effective amount of theagent of Claim 92, or agonists or antagonists thereof, therebyeffectively modulating the expression of said polypeptide.148. A method of modulating a condition associated with a disruption ofa gene which encodes for a PRO226, PRO257, PRO268, PRO290, PRO36006,PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125, PRO1134,PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433,PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341,PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737,PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873,PRO10196, PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 orPRO85161 polypeptide, the method comprising administering to a subjectwhom may have the condition, or may be prone to have the condition ormay be in whom the condition is to be prevented, a therapeuticallyeffective amount of the therapeutic agent of Claim 98, or agonists orantagonists thereof, thereby effectively modulating the condition.149. A method of treating or preventing or ameliorating a neurologicaldisorder; cardiovascular, endothelial or angiogenic disorder;immunological disorder; oncological disorder; bone metabolic abnormalityor disorder, or embryonic lethality associated with the disruption of agene which encodes for a PRO226, PRO257, PRO268, PRO290, PRO36006,PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125, PRO1134,PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433,PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341,PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737,PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873,PRO10196, PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 orPRO85161 polypeptide, the method comprising administering to a non-humantransgenic animal cell culture, each cell of said culture comprising adisruption of the gene which encodes for a PRO226, PRO257, PRO268,PRO290, PRO36006, PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071,PRO1125, PRO1134, PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387,PRO1419, PRO1433, PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904,PRO35193, PRO4341, PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985,PRO4989, PRO5737, PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821,PRO9852, PRO9873, PRO10196, PRO34778, PRO20233, PRO21956, PRO57290,PRO38465, PRO38683 or PRO85161 polypeptide, a therapeutically effectiveamount of the agent of Claim 139, or agonists or antagonists thereof,thereby effectively treating or preventing or ameliorating saiddisorder.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a nucleotide sequence (SEQ ID NO:1) of a native sequencePRO226 cDNA, wherein SEQ ID NO:1 is a clone designated herein as“DNA33460-1166” (UNQ200).

FIG. 2 shows the amino acid sequence (SEQ ID NO:2) derived from thecoding sequence of SEQ ID NO:1 shown in FIG. 1.

FIG. 3 shows a nucleotide sequence (SEQ ID NO:3) of a native sequencePRO257 cDNA, wherein SEQ ID NO:3 is a clone designated herein as“DNA35841-1173” (UNQ224).

FIG. 4 shows the amino acid sequence (SEQ ID NO:4) derived from thecoding sequence of SEQ ID NO:3 shown in FIG. 3.

FIG. 5 shows a nucleotide sequence (SEQ ID NO:5) of a native sequencePRO268 cDNA, wherein SEQ ID NO:5 is a clone designated herein as“DNA39427-1179” (UNQ235).

FIG. 6 shows the amino acid sequence (SEQ ID NO:6) derived from thecoding sequence of SEQ ID NO:5 shown in FIG. 5.

FIG. 7 shows a nucleotide sequence (SEQ ID NO: 7) of a native sequencePRO290 cDNA, wherein SEQ ID NO:7 is a clone designated herein as“DNA35680-1212” (UNQ253).

FIG. 8 shows the amino acid sequence (SEQ ID NO: 8) derived from thecoding sequence of SEQ ID NO:7 shown in FIG. 7.

FIG. 9 shows a nucleotide sequence (SEQ ID NO:9) of a native sequencePRO36006 cDNA, wherein SEQ ID NO:9 is a clone designated herein as“DNA225543” (UNQ294).

FIG. 10 shows the amino acid sequence (SEQ ID NO:10) derived from thecoding sequence of SEQ ID NO:9 shown in FIG. 9.

FIG. 11 shows a nucleotide sequence (SEQ ID NO:11) of a native sequencePRO363 cDNA, wherein SEQ ID NO:11 is a clone designated herein as“DNA45419-1252” (UNQ318).

FIG. 12 shows the amino acid sequence (SEQ ID NO:12) derived from thecoding sequence of SEQ ID NO:11 shown in FIG. 11.

FIG. 13 shows a nucleotide sequence (SEQ ID NO:13) of a native sequencePRO365 cDNA, wherein SEQ ID NO:13 is a clone designated herein as“DNA46777-1253” (UNQ320).

FIG. 14 shows the amino acid sequence (SEQ ID NO:14) derived from thecoding sequence of SEQ ID NO:13 shown in FIG. 13.

FIG. 15 shows a nucleotide sequence (SEQ ID NO:15) of a native sequencePRO382 cDNA, wherein SEQ ID NO:15 is a clone designated herein as“DNA45234-1277” (UNQ323).

FIG. 16 shows the amino acid sequence (SEQ ID NO:16) derived from thecoding sequence of SEQ ID NO:15 shown in FIG. 15.

FIG. 17 shows a nucleotide sequence (SEQ ID NO:17) of a native sequencePRO444 cDNA, wherein SEQ ID NO:17 is a clone designated herein as“DNA26846-1397” (UNQ328).

FIG. 18 shows the amino acid sequence (SEQ ID NO:18) derived from thecoding sequence of SEQ ID NO:17 shown in FIG. 17.

FIG. 19 shows a nucleotide sequence (SEQ ID NO:19) of a native sequencePRO705 cDNA, wherein SEQ ID NO:19 is a clone designated herein as“DNA50914-1289” (UNQ369).

FIG. 20 shows the amino acid sequence (SEQ ID NO:20) derived from thecoding sequence of SEQ ID NO:19 shown in FIG. 19.

FIG. 21 shows a nucleotide sequence (SEQ ID NO:21) of a native sequencePRO1071 cDNA, wherein SEQ ID NO:21 is a clone designated herein as“DNA58847-1383” (UNQ528).

FIG. 22 shows the amino acid sequence (SEQ ID NO:22) derived from thecoding sequence of SEQ ID NO:21 shown in FIG. 21.

FIG. 23 shows a nucleotide sequence (SEQ ID NO:23) of a native sequencePRO1125 cDNA, wherein SEQ ID NO:23 is a clone designated herein as“DNA60619-1482” (UNQ563).

FIG. 24 shows the amino acid sequence (SEQ ID NO:24) derived from thecoding sequence of SEQ ID NO:23 shown in FIG. 23.

FIG. 25 shows a nucleotide sequence (SEQ ID NO:25) of a native sequencePRO1134 cDNA, wherein SEQ ID NO:25 is a clone designated herein as“DNA56865-1491” (UNQ572).

FIG. 26 shows the amino acid sequence (SEQ ID NO:26) derived from thecoding sequence of SEQ ID NO:25 shown in FIG. 25.

FIG. 27 shows a nucleotide sequence (SEQ ID NO:27) of a native sequencePRO1155 cDNA, wherein SEQ ID NO:27 is a clone designated herein as“DNA59849-1504” (UNQ585).

FIG. 28 shows the amino acid sequence (SEQ ID NO:28) derived from thecoding sequence of SEQ ID NO:27 shown in FIG. 27.

FIG. 29 shows a nucleotide sequence (SEQ ID NO:29) of a native sequencePRO1281 cDNA, wherein SEQ ID NO:29 is a clone designated herein as“DNA59820-1549” (UNQ651).

FIG. 30 shows the amino acid sequence (SEQ ID NO:30) derived from thecoding sequence of SEQ ID NO:29 shown in FIG. 29.

FIG. 31 shows a nucleotide sequence (SEQ ID NO:31) of a native sequencePRO1343 cDNA, wherein SEQ ID NO:31 is a clone designated herein as“DNA66675-1587” (UNQ698).

FIG. 32 shows the amino acid sequence (SEQ ID NO:32) derived from thecoding sequence of SEQ ID NO:31 shown in FIG. 31.

FIG. 33 shows a nucleotide sequence (SEQ ID NO:33) of a native sequencePRO1379 cDNA, wherein SEQ ID NO:33 is a clone designated herein as“DNA59828-1608” (UNQ716).

FIG. 34 shows the amino acid sequence (SEQ ID NO:34) derived from thecoding sequence of SEQ ID NO:33 shown in FIG. 33.

FIG. 35 shows a nucleotide sequence (SEQ ID NO:35) of a native sequencePRO1380 cDNA, wherein SEQ ID NO:35 is a clone designated herein as“DNA60740-1615” (UNQ717).

FIG. 36 shows the amino acid sequence (SEQ ID NO: 36) derived from thecoding sequence of SEQ ID NO:35 shown in FIG. 35.

FIG. 37 shows a nucleotide sequence (SEQ ID NO:37) of a native sequencePRO1387 cDNA, wherein SEQ ID NO:37 is a clone designated herein as“DNA68872-1620” (UNQ722).

FIG. 38 shows the amino acid sequence (SEQ ID NO: 38) derived from thecoding sequence of SEQ ID NO:37 shown in FIG. 37.

FIG. 39 shows a nucleotide sequence (SEQ ID NO:39) of a native sequencePRO1419 cDNA, wherein SEQ ID NO:39 is a clone designated herein as“DNA71290-1630” (UNQ733).

FIG. 40 shows the amino acid sequence (SEQ ID NO:40) derived from thecoding sequence of SEQ ID NO:39 shown in FIG. 39.

FIG. 41 shows a nucleotide sequence (SEQ ID NO:41) of a native sequencePRO1433 cDNA, wherein SEQ ID NO:41 is a clone designated herein as“DNA71184-1634” (UNQ738).

FIG. 42 shows the amino acid sequence (SEQ ID NO:42) derived from thecoding sequence of SEQ ID NO:41 shown in FIG. 41.

FIG. 43 shows a nucleotide sequence (SEQ ID NO:43) of a native sequencePRO1474 cDNA, wherein SEQ ID NO:43 is a clone designated herein as“DNA73739-1645” (UNQ745).

FIG. 44 shows the amino acid sequence (SEQ ID NO:44) derived from thecoding sequence of SEQ ID NO:43 shown in FIG. 43.

FIG. 45 shows a nucleotide sequence (SEQ ID NO:45) of a native sequencePRO1550 cDNA, wherein SEQ ID NO:45 is a clone designated herein as“DNA76393-1664” (UNQ762).

FIG. 46 shows the amino acid sequence (SEQ ID NO:46) derived from thecoding sequence of SEQ ID NO:45 shown in FIG. 45.

FIG. 47 shows a nucleotide sequence (SEQ ID NO:47) of a native sequencePRO1571 cDNA, wherein SEQ ID NO:47 is a clone designated herein as“DNA73730-1679” (UNQ777).

FIG. 48 shows the amino acid sequence (SEQ ID NO:48) derived from thecoding sequence of SEQ ID NO:47 shown in FIG. 47.

FIG. 49 shows a nucleotide sequence (SEQ ID NO:49) of a native sequencePRO1572 cDNA, wherein SEQ ID NO:49 is a clone designated herein as“DNA73734-1680” (UNQ778).

FIG. 50 shows the amino acid sequence (SEQ ID NO: 50) derived from thecoding sequence of SEQ ID NO:49 shown in FIG. 49.

FIG. 51 shows a nucleotide sequence (SEQ ID NO:51) of a native sequencePRO1759 cDNA, wherein SEQ ID NO:51 is a clone designated herein as“DNA76531-1701” (UNQ832).

FIG. 52 shows the amino acid sequence (SEQ ID NO: 52) derived from thecoding sequence of SEQ ID NO:51 shown in FIG. 51.

FIG. 53 shows a nucleotide sequence (SEQ ID NO:53) of a native sequencePRO1904 cDNA, wherein SEQ ID NO:53 is a clone designated herein as“DNA82372” (UNQ886).

FIG. 54 shows the amino acid sequence (SEQ ID NO: 54) derived from thecoding sequence of SEQ ID NO:53 shown in FIG. 53.

FIG. 55 shows a nucleotide sequence (SEQ ID NO:55) of a native sequencePRO35193 cDNA, wherein SEQ ID NO:55 is a clone designated herein as“DNA225681” (UNQ983).

FIG. 56 shows the amino acid sequence (SEQ ID NO:56) derived from thecoding sequence of SEQ ID NO:55 shown in FIG. 55.

FIG. 57 shows a nucleotide sequence (SEQ ID NO:57) of a native sequencePRO4341 cDNA, wherein SEQ ID NO:57 is a clone designated herein as“DNA81761-2583” (UNQ1895).

FIG. 58 shows the amino acid sequence (SEQ ID NO:58) derived from thecoding sequence of SEQ ID NO:57 shown in FIG. 57.

FIG. 59 shows a nucleotide sequence (SEQ ID NO:59) of a native sequencePRO4348 cDNA, wherein SEQ ID NO:59 is a clone designated herein as“DNA92232-2589” (UNQ1902).

FIG. 60 shows the amino acid sequence (SEQ ID NO:60) derived from thecoding sequence of SEQ ID NO:59 shown in FIG. 59.

FIG. 61 shows a nucleotide sequence (SEQ ID NO:61) of a native sequencePRO4369 cDNA, wherein SEQ ID NO:61 is a clone designated herein as“DNA92289-2598” (UNQ1911).

FIG. 62 shows the amino acid sequence (SEQ ID NO: 62) derived from thecoding sequence of SEQ ID NO:61 shown in FIG. 61.

FIG. 63 shows a nucleotide sequence (SEQ ID NO:63) of a native sequencePRO4381 cDNA, wherein SEQ ID NO:63 is a clone designated herein as“DNA92225-2603” (UNQ1916).

FIG. 64 shows the amino acid sequence (SEQ ID NO: 64) derived from thecoding sequence of SEQ ID NO:63 shown in FIG. 63.

FIG. 65 shows a nucleotide sequence (SEQ ID NO:65) of a native sequencePRO4407 cDNA, wherein SEQ ID NO:65 is a clone designated herein as“DNA92264-2616” (UNQ1932).

FIG. 66 shows the amino acid sequence (SEQ ID NO: 66) derived from thecoding sequence of SEQ ID NO:65 shown in FIG. 65.

FIG. 67 shows a nucleotide sequence (SEQ ID NO:67) of a native sequencePRO4425 cDNA, wherein SEQ ID NO:67 is a clone designated herein as“DNA93011-2637” (UNQ1942).

FIG. 68 shows the amino acid sequence (SEQ ID NO:68) derived from thecoding sequence of SEQ ID NO:67 shown in FIG. 67.

FIG. 69 shows a nucleotide sequence (SEQ ID NO:69) of a native sequencePRO4985 cDNA, wherein SEQ ID NO:69 is a clone designated herein as“DNA59770-2652” (UNQ2426).

FIG. 70 shows the amino acid sequence (SEQ ID NO: 70) derived from thecoding sequence of SEQ ID NO:69 shown in FIG. 69.

FIG. 71 shows a nucleotide sequence (SEQ ID NO:71) of a native sequencePRO4989 cDNA, wherein SEQ ID NO:71 is a clone designated herein as“DNA80135-2655” (UNQ2429).

FIG. 72 shows the amino acid sequence (SEQ ID NO: 72) derived from thecoding sequence of SEQ ID NO:71 shown in FIG. 71.

FIG. 73 shows a nucleotide sequence (SEQ ID NO:73) of a native sequencePRO5737 cDNA, wherein SEQ ID NO:73 is a clone designated herein as“DNA92929-2534-1” (UNQ2456).

FIG. 74 shows the amino acid sequence (SEQ ID NO:74) derived from thecoding sequence of SEQ ID NO:73 shown in FIG. 73.

FIG. 75 shows a nucleotide sequence (SEQ ID NO:75) of a native sequencePRO5800 cDNA, wherein SEQ ID NO:75 is a clone designated herein as“DNA108912-2680” (UNQ2500).

FIG. 76 shows the amino acid sequence (SEQ ID NO: 76) derived from thecoding sequence of SEQ ID NO:75 shown in FIG. 75.

FIG. 77 shows a nucleotide sequence (SEQ ID NO:77) of a native sequencePRO5993 cDNA, wherein SEQ ID NO:77 is a clone designated herein as“DNA100276-2684” (UNQ2504).

FIG. 78 shows the amino acid sequence (SEQ ID NO:78) derived from thecoding sequence of SEQ ID NO:77 shown in FIG. 77.

FIG. 79 shows a nucleotide sequence (SEQ ID NO:79) of a native sequencePRO6017 cDNA, wherein SEQ ID NO:79 is a clone designated herein as“DNA96860-2700” (UNQ2524).

FIG. 80 shows the amino acid sequence (SEQ ID NO: 80) derived from thecoding sequence of SEQ ID NO:79 shown in FIG. 79.

FIG. 81 shows a nucleotide sequence (SEQ ID NO: 81) of a native sequencePRO7174 cDNA, wherein SEQ ID NO:81 is a clone designated herein as“DNA96883-2745” (UNQ2784).

FIG. 82 shows the amino acid sequence (SEQ ID NO: 82) derived from thecoding sequence of SEQ ID NO:81 shown in FIG. 81.

FIG. 83 shows a nucleotide sequence (SEQ ID NO:83) of a native sequencePRO9744 cDNA, wherein SEQ ID NO:83 is a clone designated herein as“DNA136110-2763” (UNQ3003).

FIG. 84 shows the amino acid sequence (SEQ ID NO: 84) derived from thecoding sequence of SEQ ID NO:83 shown in FIG. 83.

FIG. 85 shows a nucleotide sequence (SEQ ID NO:85) of a native sequencePRO9821 cDNA, wherein SEQ ID NO:85 is a clone designated herein as“DNA108725-2766” (UNQ3023).

FIG. 86 shows the amino acid sequence (SEQ ID NO: 86) derived from thecoding sequence of SEQ ID NO:85 shown in FIG. 85.

FIG. 87 shows a nucleotide sequence (SEQ ID NO:87) of a native sequencePRO9852 cDNA, wherein SEQ ID NO:87 is a clone designated herein as“DNA129332-2775” (UNQ3037).

FIG. 88 shows the amino acid sequence (SEQ ID NO:88) derived from thecoding sequence of SEQ ID NO:87 shown in FIG. 87.

FIG. 89 shows a nucleotide sequence (SEQ ID NO: 89) of a native sequencePRO9873 cDNA, wherein SEQ ID NO: 89 is a clone designated herein as“DNA143076-2787” (UNQ3054).

FIG. 90 shows the amino acid sequence (SEQ ID NO:90) derived from thecoding sequence of SEQ ID NO:89 shown in FIG. 89.

FIG. 91 shows a nucleotide sequence (SEQ ID NO:91) of a native sequencePRO10196 cDNA, wherein SEQ ID NO:91 is a clone designated herein as“DNA144841-2816” (UNQ3115).

FIG. 92 shows the amino acid sequence (SEQ ID NO:92) derived from thecoding sequence of SEQ ID NO:91 shown in FIG. 91.

FIG. 93 shows a nucleotide sequence (SEQ ID NO:93) of a native sequencePRO34778 cDNA, wherein SEQ ID NO:93 is a clone designated herein as“DNA220432” (UNQ3966).

FIG. 94 shows the amino acid sequence (SEQ ID NO:94) derived from thecoding sequence of SEQ ID NO:93 shown in FIG. 93.

FIG. 95 shows a nucleotide sequence (SEQ ID NO:95) of a native sequencePRO20233 cDNA, wherein SEQ ID NO:95 is a clone designated herein as“DNA165608” (UNQ6208).

FIG. 96 shows the amino acid sequence (SEQ ID NO:96) derived from thecoding sequence of SEQ ID NO:95 shown in FIG. 95.

FIG. 97 shows a nucleotide sequence (SEQ ID NO:97) of a native sequencePRO21956 cDNA, wherein SEQ ID NO:97 is a clone designated herein as“DNA178511-2986” (UNQ6973).

FIG. 98 shows the amino acid sequence (SEQ ID NO:98) derived from thecoding sequence of SEQ ID NO:97 shown in FIG. 97.

FIG. 99 shows a nucleotide sequence (SEQ ID NO:99) of a native sequencePRO57290 cDNA, wherein SEQ ID NO:99 is a clone designated herein as“DNA269238” (UNQ8782).

FIG. 100 shows the amino acid sequence (SEQ ID NO:100) derived from thecoding sequence of SEQ ID NO:99 shown in FIG. 99.

FIG. 101 shows a nucleotide sequence (SEQ ID NO:101) of a nativesequence PRO38465 cDNA, wherein SEQ ID NO:101 is a clone designatedherein as “DNA228002” (UNQ9128).

FIG. 102 shows the amino acid sequence (SEQ ID NO:102) derived from thecoding sequence of SEQ ID NO:101 shown in FIG. 101.

FIG. 103 shows a nucleotide sequence (SEQ ID NO:103) of a nativesequence PRO38683 cDNA, wherein SEQ ID NO:103 is a clone designatedherein as “DNA228199” (UNQ9638).

FIG. 104 shows the amino acid sequence (SEQ ID NO:104) derived from thecoding sequence of SEQ ID NO:103 shown in FIG. 103.

FIG. 105 shows a nucleotide sequence (SEQ ID NO:105) of a nativesequence PRO85161 cDNA, wherein SEQ ID NO:105 is a clone designatedherein as “DNA329632” (UNQ16168).

FIG. 106 shows the amino acid sequence (SEQ ID NO:106) derived from thecoding sequence of SEQ ID NO:105 shown in FIG. 105.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS I. Definitions

The terms “PRO polypeptide” and “PRO” as used herein and whenimmediately followed by a numerical designation refer to variouspolypeptides, wherein the complete designation (i.e., PRO/number) refersto specific polypeptide sequences as described herein. The terms“PRO/number polypeptide” and “PRO/number” wherein the term “number” isprovided as an actual numerical designation as used herein encompassnative sequence polypeptides and polypeptide variants (which are furtherdefined herein). The PRO226, PRO257, PRO268, PRO290, PRO36006, PRO363,PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155,PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474,PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348,PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800,PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196,PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161polypeptides described herein may be isolated from a variety of sources,such as from human tissue types or from another source, or prepared byrecombinant or synthetic methods. The term “PRO polypeptide” refers toeach individual PRO/number polypeptide disclosed herein. All disclosuresin this specification which refer to the “PRO polypeptide” refer to eachof the polypeptides individually as well as jointly. For example,descriptions of the preparation of, purification of, derivation of,formation of antibodies to or against, administration of, compositionscontaining, treatment of a disease with, etc., pertain to eachpolypeptide of the invention individually. The term “PRO polypeptide”also includes variants of the PRO/number polypeptides disclosed herein.

A “native sequence PRO226, PRO257, PRO268, PRO290, PRO36006, PRO363,PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155,PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474,PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348,PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800,PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196,PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161polypeptide” comprises a polypeptide having the same amino acid sequenceas the corresponding PRO226, PRO257, PRO268, PRO290, PRO36006, PRO363,PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155,PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474,PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348,PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800,PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196,PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161polypeptide derived from nature. Such native sequence PRO226, PRO257,PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382, PRO444, PRO705,PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343, PRO1379, PRO1380,PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571, PRO1572, PRO1759,PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381, PRO4407, PRO4425,PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017, PRO7174, PRO9744,PRO9821, PRO9852, PRO9873, PRO10196, PRO34778, PRO20233, PRO21956,PRO57290, PRO38465, PRO38683 or PRO85161 polypeptides can be isolatedfrom nature or can be produced by recombinant or synthetic means. Theterm “native sequence PRO226, PRO257, PRO268, PRO290, PRO36006, PRO363,PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155,PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474,PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348,PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800,PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196,PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161polypeptide” specifically encompasses naturally-occurring truncated orsecreted forms of the specific PRO226, PRO257, PRO268, PRO290, PRO36006,PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125, PRO1134,PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433,PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341,PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737,PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873,PRO10196, PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 orPRO85161 polypeptide (e.g., an extracellular domain sequence),naturally-occurring variant forms (e.g., alternatively spliced forms)and naturally-occurring allelic variants of the polypeptide. Theinvention provides native sequence PRO226, PRO257, PRO268, PRO290,PRO36006, PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125,PRO1134, PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419,PRO1433, PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193,PRO4341, PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989,PRO5737, PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852,PRO9873, PRO10196, PRO34778, PRO20233, PRO21956, PRO57290, PRO38465,PRO38683 or PRO85161 polypeptides disclosed herein which are mature orfull-length native sequence polypeptides comprising the full-lengthamino acids sequences shown in the accompanying figures. Start and stopcodons are shown in bold font and underlined in the figures. However,while the PRO226, PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365,PRO382, PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281,PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550,PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369,PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993,PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196,PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161polypeptide disclosed in the accompanying figures are shown to beginwith methionine residues designated herein as amino acid position 1 inthe figures, it is conceivable and possible that other methionineresidues located either upstream or downstream from the amino acidposition 1 in the figures may be employed as the starting amino acidresidue for the PRO226, PRO257, PRO268, PRO290, PRO36006, PRO363,PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155,PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474,PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348,PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800,PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196,PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161polypeptides.

The PRO226, PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382,PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343,PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571,PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381,PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017,PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196, PRO34778,PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161 polypeptide“extracellular domain” or “ECD” refers to a form of the PRO226, PRO257,PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382, PRO444, PRO705,PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343, PRO1379, PRO1380,PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571, PRO1572, PRO1759,PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381, PRO4407, PRO4425,PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017, PRO7174, PRO9744,PRO9821, PRO9852, PRO9873, PRO10196, PRO34778, PRO20233, PRO21956,PRO57290, PRO38465, PRO38683 or PRO85161 polypeptide which isessentially free of the transmembrane and cytoplasmic domains.Ordinarily, a PRO226, PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365,PRO382, PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281,PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550,PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369,PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993,PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196,PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161polypeptide ECD will have less than 1% of such transmembrane and/orcytoplasmic domains and preferably, will have less than 0.5% of suchdomains. It will be understood that any transmembrane domains identifiedfor the PRO226, PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365,PRO382, PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281,PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550,PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369,PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993,PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196,PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161polypeptides of the present invention are identified pursuant tocriteria routinely employed in the art for identifying that type ofhydrophobic domain. The exact boundaries of a transmembrane domain mayvary but most likely by no more than about 5 amino acids at either endof the domain as initially identified herein. Optionally, therefore, anextracellular domain of a PRO226, PRO257, PRO268, PRO290, PRO36006,PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125, PRO1134,PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433,PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341,PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737,PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873,PRO10196, PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 orPRO85161 polypeptide may contain from about 5 or fewer amino acids oneither side of the transmembrane domain/extracellular domain boundary asidentified in the Examples or specification and such polypeptides, withor without the associated signal peptide, and nucleic acid encodingthem, are contemplated by the present invention.

The approximate location of the “signal peptides” of the various PRO226,PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382, PRO444,PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343, PRO1379,PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571, PRO1572,PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381, PRO4407,PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017, PRO7174,PRO9744, PRO9821, PRO9852, PRO9873, PRO10196, PRO34778, PRO20233,PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161 polypeptidesdisclosed herein are shown in the present specification and/or theaccompanying figures. It is noted, however, that the C-terminal boundaryof a signal peptide may vary, but most likely by no more than about 5amino acids on either side of the signal peptide C-terminal boundary asinitially identified herein, wherein the C-terminal boundary of thesignal peptide may be identified pursuant to criteria routinely employedin the art for identifying that type of amino acid sequence element(e.g., Nielsen et al., Prot. Eng. 10:1-6 (1997) and von Heinje et al.,Nucl. Acids. Res. 14:4683-4690 (1986)). Moreover, it is also recognizedthat, in some cases, cleavage of a signal sequence from a secretedpolypeptide is not entirely uniform, resulting in more than one secretedspecies. These mature polypeptides, where the signal peptide is cleavedwithin no more than about 5 amino acids on either side of the C-terminalboundary of the signal peptide as identified herein, and thepolynucleotides encoding them, are contemplated by the presentinvention.

“PRO226, PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382,PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343,PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571,PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381,PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017,PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196, PRO34778,PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161 polypeptidevariant” means a PRO226, PRO257, PRO268, PRO290, PRO36006, PRO363,PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO155,PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474,PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348,PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800,PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196,PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161polypeptide, preferably an active PRO226, PRO257, PRO268, PRO290,PRO36006, PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125,PRO1134, PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419,PRO1433, PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193,PRO4341, PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989,PRO5737, PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852,PRO9873, PRO10196, PRO34778, PRO20233, PRO21956, PRO57290, PRO38465,PRO38683 or PRO85161 polypeptide, as defined herein having at leastabout 80% amino acid sequence identity with a full-length nativesequence PRO226, PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365,PRO382, PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281,PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550,PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369,PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993,PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196,PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161polypeptide sequence as disclosed herein, a PRO226, PRO257, PRO268,PRO290, PRO36006, PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071,PRO1125, PRO1134, PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387,PRO1419, PRO1433, PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904,PRO35193, PRO4341, PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985,PRO4989, PRO5737, PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821,PRO9852, PRO9873, PRO10196, PRO34778, PRO20233, PRO21956, PRO57290,PRO38465, PRO38683 or PRO85161 polypeptide sequence lacking the signalpeptide as disclosed herein, an extracellular domain of a PRO226,PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382, PRO444,PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343, PRO1379,PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571, PRO1572,PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381, PRO4407,PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017, PRO7174,PRO9744, PRO9821, PRO9852, PRO9873, PRO10196, PRO34778, PRO20233,PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161 polypeptide, with orwithout the signal peptide, as disclosed herein or any other fragment ofa full-length PRO226, PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365,PRO382, PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281,PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550,PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369,PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993,PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196,PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161polypeptide sequence as disclosed herein (such as those encoded by anucleic acid that represents only a portion of the complete codingsequence for a full-length PRO226, PRO257, PRO268, PRO290, PRO36006,PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125, PRO1134,PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433,PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341,PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737,PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873,PRO10196, PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 orPRO85161 polypeptide). Such PRO226, PRO257, PRO268, PRO290, PRO36006,PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125, PRO1134,PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433,PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341,PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737,PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873,PRO10196, PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 orPRO85161 polypeptide variants include, for instance, PRO226, PRO257,PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382, PRO444, PRO705,PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343, PRO1379, PRO1380,PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571, PRO1572, PRO1759,PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381, PRO4407, PRO4425,PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017, PRO7174, PRO9744,PRO9821, PRO9852, PRO9873, PRO10196, PRO34778, PRO20233, PRO21956,PRO57290, PRO38465, PRO38683 or PRO85161 polypeptides wherein one ormore amino acid residues are added, or deleted, at the N or C-terminusof the full-length native amino acid sequence. Ordinarily, a PRO226,PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382, PRO444,PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343, PRO1379,PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571, PRO1572,PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381, PRO4407,PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017, PRO7174,PRO9744, PRO9821, PRO9852, PRO9873, PRO10196, PRO34778, PRO20233,PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161 polypeptide variantwill have or will have at least about 80% amino acid sequence identity,alternatively will have or will have at least about 81%, 82%, 83%, 84%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99% amino acid sequence identity, to a full-length native sequencePRO226, PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382,PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343,PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571,PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381,PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017,PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196, PRO34778,PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161 polypeptidesequence as disclosed herein, a PRO226, PRO257, PRO268, PRO290,PRO36006, PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125,PRO1134, PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419,PRO1433, PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193,PRO4341, PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989,PRO5737, PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852,PRO9873, PRO10196, PRO34778, PRO20233, PRO21956, PRO57290, PRO38465,PRO38683 or PRO85161 polypeptide sequence lacking the signal peptide asdisclosed herein, an extracellular domain of a PRO226, PRO257, PRO268,PRO290, PRO36006, PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071,PRO1125, PRO1134, PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387,PRO1419, PRO1433, PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904,PRO35193, PRO4341, PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985,PRO4989, PRO5737, PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821,PRO9852, PRO9873, PRO10196, PRO34778, PRO20233, PRO21956, PRO57290,PRO38465, PRO38683 or PRO85161 polypeptide, with or without the signalpeptide, as disclosed herein or any other specifically defined fragmentof a full-length PRO226, PRO257, PRO268, PRO290, PRO36006, PRO363,PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155,PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474,PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348,PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800,PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196,PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161polypeptide sequence as disclosed herein. Ordinarily, PRO226, PRO257,PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382, PRO444, PRO705,PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343, PRO1379, PRO1380,PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571, PRO1572, PRO1759,PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381, PRO4407, PRO4425,PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017, PRO7174, PRO9744,PRO9821, PRO9852, PRO9873, PRO10196, PRO34778, PRO20233, PRO21956,PRO57290, PRO38465, PRO38683 or PRO85161 variant polypeptides are or areat least about 10 amino acids in length, alternatively are or are atleast about 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140,150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280,290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420,430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560,570, 580, 590, 600 amino acids in length, or more. Optionally, PRO226,PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382, PRO444,PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343, PRO1379,PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571, PRO1572,PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381, PRO4407,PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017, PRO7174,PRO9744, PRO9821, PRO9852, PRO9873, PRO10196, PRO34778, PRO20233,PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161 variant polypeptideswill have no more than one conservative amino acid substitution ascompared to the native PRO226, PRO257, PRO268, PRO290, PRO36006, PRO363,PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155,PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474,PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348,PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800,PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196,PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161polypeptide sequence, alternatively will have or will have no more than2, 3, 4, 5, 6, 7, 8, 9, or 10 conservative amino acid substitution ascompared to the native PRO226, PRO257, PRO268, PRO290, PRO36006, PRO363,PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155,PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474,PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348,PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800,PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196,PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161polypeptide sequence.

“Percent (%) amino acid sequence identity” with respect to the PRO226,PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382, PRO444,PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343, PRO1379,PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571, PRO1572,PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381, PRO4407,PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017, PRO7174,PRO9744, PRO9821, PRO9852, PRO9873, PRO10196, PRO34778, PRO20233,PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161 polypeptide sequencesidentified herein is defined as the percentage of amino acid residues ina candidate sequence that are identical with the amino acid residues inthe specific PRO226, PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365,PRO382, PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281,PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550,PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369,PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993,PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196,PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161polypeptide sequence, after aligning the sequences and introducing gaps,if necessary, to achieve the maximum percent sequence identity, and notconsidering any conservative substitutions as part of the sequenceidentity. Alignment for purposes of determining percent amino acidsequence identity can be achieved in various ways that are within theskill in the art, for instance, using publicly available computersoftware such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software.Those skilled in the art can determine appropriate parameters formeasuring alignment, including any algorithms needed to achieve maximalalignment over the full length of the sequences being compared. Forpurposes herein, however, % amino acid sequence identity values aregenerated using the sequence comparison computer program ALIGN-2,wherein the complete source code for the ALIGN-2 program is provided inTable 1 below. The ALIGN-2 sequence comparison computer program wasauthored by Genentech, Inc. and the source code shown in Table 1 belowhas been filed with user documentation in the U.S. Copyright Office,Washington D.C., 20559, where it is registered under U.S. CopyrightRegistration No. TXU510087. The ALIGN-2 program is publicly availablethrough Genentech, Inc., South San Francisco, Calif. or may be compiledfrom the source code provided in Table 1 below. The ALIGN-2 programshould be compiled for use on a UNIX operating system, preferablydigital UNIX V4.0D. All sequence comparison parameters are set by theALIGN-2 program and do not vary.

In situations where ALIGN-2 is employed for amino acid sequencecomparisons, the % amino acid sequence identity of a given amino acidsequence A to, with, or against a given amino acid sequence B (which canalternatively be phrased as a given amino acid sequence A that has orcomprises a certain % amino acid sequence identity to, with, or againsta given amino acid sequence B) is calculated as follows:100 times the fraction X/Ywhere X is the number of amino acid residues scored as identical matchesby the sequence alignment program ALIGN-2 in that program's alignment ofA and B, and where Y is the total number of amino acid residues in B. Itwill be appreciated that where the length of amino acid sequence A isnot equal to the length of amino acid sequence B, the % amino acidsequence identity of A to B will not equal the % amino acid sequenceidentity of B to A. As examples of % amino acid sequence identitycalculations using this method, Tables 2 and 3 demonstrate how tocalculate the % amino acid sequence identity of the amino acid sequencedesignated “Comparison Protein” to the amino acid sequence designated“PRO”, wherein “PRO” represents the amino acid sequence of ahypothetical PRO polypeptide of interest, “Comparison Protein”represents the amino acid sequence of a polypeptide against which the“PRO” polypeptide of interest is being compared, and “X, “Y” and “Z”each represent different hypothetical amino acid residues. Unlessspecifically stated otherwise, all % amino acid sequence identity valuesused herein are obtained as described in the immediately precedingparagraph using the ALIGN-2 computer program.

“PRO226, PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382,PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343,PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571,PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381,PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO617,PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196, PRO34778,PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161 variantpolynucleotide” or “PRO226, PRO257, PRO268, PRO290, PRO36006, PRO363,PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155,PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474,PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348,PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800,PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196,PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161variant nucleic acid sequence” means a nucleic acid molecule whichencodes a PRO226, PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365,PRO382, PRO444, PRO705, PRO1071, PRO125, PRO1134, PRO1155, PRO1281,PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550,PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369,PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993,PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196,PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161polypeptide, preferably an active PRO226, PRO257, PRO268, PRO290,PRO36006, PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125,PRO1134, PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419,PRO1433, PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193,PRO4341, PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989,PRO5737, PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852,PRO9873, PRO1196, PRO34778, PRO20233, PRO21956, PRO57290, PRO38465,PRO38683 or PRO85161 polypeptide, as defined herein and which has atleast about 80% nucleic acid sequence identity with a nucleotide acidsequence encoding a full-length native sequence PRO226, PRO257, PRO268,PRO290, PRO36006, PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071,PRO1125, PRO1134, PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387,PRO1419, PRO1433, PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904,PRO35193, PRO4341, PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985,PRO4989, PRO5737, PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821,PRO9852, PRO9873, PRO10196, PRO34778, PRO20233, PRO21956, PRO57290,PRO38465, PRO38683 or PRO85161 polypeptide sequence as disclosed herein,a full-length native sequence PRO226, PRO257, PRO268, PRO290, PRO36006,PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125, PRO1134,PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433,PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341,PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737,PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873,PRO10196, PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 orPRO85161 polypeptide sequence lacking the signal peptide as disclosedherein, an extracellular domain of a PRO226, PRO257, PRO268, PRO290,PRO36006, PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125,PRO1134, PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419,PRO1433, PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193,PRO4341, PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989,PRO5737, PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852,PRO9873, PRO10196, PRO34778, PRO20233, PRO21956, PRO57290, PRO38465,PRO38683 or PRO85161 polypeptide, with or without the signal peptide, asdisclosed herein or any other fragment of a full-length PRO226, PRO257,PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382, PRO444, PRO705,PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343, PRO1379, PRO1380,PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571, PRO1572, PRO1759,PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381, PRO4407, PRO4425,PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017, PRO7174, PRO9744,PRO9821, PRO9852, PRO9873, PRO10196, PRO34778, PRO20233, PRO21956,PRO57290, PRO38465, PRO38683 or PRO85161 polypeptide sequence asdisclosed herein (such as those encoded by a nucleic acid thatrepresents only a portion of the complete coding sequence for afull-length PRO226, PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365,PRO382, PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281,PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550,PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369,PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993,PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196,PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161polypeptide). Ordinarily, a PRO226, PRO257, PRO268, PRO290, PRO36006,PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125, PRO1134,PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433,PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341,PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737,PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873,PRO10196, PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 orPRO85161 variant polynucleotide will have or will have at least about80% nucleic acid sequence identity, alternatively will have or will haveat least about 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% nucleic acid sequence identitywith a nucleic acid sequence encoding a full-length native sequencePRO226, PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382,PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343,PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571,PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381,PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017,PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196, PRO34778,PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161 polypeptidesequence as disclosed herein, a full-length native sequence PRO226,PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382, PRO444,PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343, PRO1379,PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571, PRO1572,PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381, PRO4407,PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017, PRO7174,PRO9744, PRO9821, PRO9852, PRO9873, PRO10196, PRO34778, PRO20233,PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161 polypeptide sequencelacking the signal peptide as disclosed herein, an extracellular domainof a PRO226, PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382,PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343,PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571,PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381,PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017,PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196, PRO34778,PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161polypeptide, with or without the signal sequence, as disclosed herein orany other fragment of a full-length PRO226, PRO257, PRO268, PRO290,PRO36006, PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125,PRO1134, PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419,PRO1433, PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193,PRO4341, PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989,PRO5737, PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852,PRO9873, PRO10196, PRO34778, PRO20233, PRO21956, PRO57290, PRO38465,PRO38683 or PRO85161 polypeptide sequence as disclosed herein. Variantsdo not encompass the native nucleotide sequence.

Ordinarily, PRO226, PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365,PRO382, PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281,PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550,PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369,PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993,PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196,PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161variant polynucleotides are or are at least about 5 nucleotides inlength, alternatively are or are at least about 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115,120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185,190, 195, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310,320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450,460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590,600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730,740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870,880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, or 1000nucleotides in length, wherein in this context the term “about” meansthe referenced nucleotide sequence length plus or minus 10% of thatreferenced length.

“Percent (%) nucleic acid sequence identity” with respect to PRO226-,PRO257-, PRO268-, PRO290-, PRO36006-, PRO363-, PRO365-, PRO382-,PRO444-, PRO705-, PRO1071-, PRO1125-, PRO1134-, PRO1155-, PRO1281-,PRO1343-, PRO1379-, PRO1380-, PRO1387-, PRO1419-, PRO1433-, PRO1474-,PRO1550-, PRO1571-, PRO1572-, PRO1759-, PRO1904-, PRO35193-, PRO4341-,PRO4348-, PRO4369-, PRO4381-, PRO4407-, PRO4425-, PRO4985-, PRO4989-,PRO5737-, PRO5800-, PRO5993-, PRO6017-, PRO7174-, PRO9744-, PRO9821-,PRO9852-, PRO9873-, PRO10196-, PRO34778-, PRO20233-, PRO21956-,PRO57290-, PRO38465-, PRO38683- or PRO85161-encoding nucleic acidsequences identified herein is defined as the percentage of nucleotidesin a candidate sequence that are identical with the nucleotides in thePRO226, PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382,PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343,PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571,PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381,PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017,PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196, PRO34778,PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161 nucleicacid sequence of interest, after aligning the sequences and introducinggaps, if necessary, to achieve the maximum percent sequence identity.Alignment for purposes of determining percent nucleic acid sequenceidentity can be achieved in various ways that are within the skill inthe art, for instance, using publicly available computer software suchas BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. For purposesherein, however, % nucleic acid sequence identity values are generatedusing the sequence comparison computer program ALIGN-2, wherein thecomplete source code for the ALIGN-2 program is provided in Table 1below. The ALIGN-2 sequence comparison computer program was authored byGenentech, Inc. and the source code shown in Table 1 below has beenfiled with user documentation in the U.S. Copyright Office, WashingtonD.C., 20559, where it is registered under U.S. Copyright RegistrationNo. TXU510087. The ALIGN-2 program is publicly available throughGenentech, Inc., South San Francisco, Calif. or may be compiled from thesource code provided in Table 1 below. The ALIGN-2 program should becompiled for use on a UNIX operating system, preferably digital UNIXV4.0D. All sequence comparison parameters are set by the ALIGN-2 programand do not vary.

In situations where ALIGN-2 is employed for nucleic acid sequencecomparisons, the % nucleic acid sequence identity of a given nucleicacid sequence C to, with, or against a given nucleic acid sequence D(which can alternatively be phrased as a given nucleic acid sequence Cthat has or comprises a certain % nucleic acid sequence identity to,with, or against a given nucleic acid sequence D) is calculated asfollows:100 times the fraction W/Zwhere W is the number of nucleotides scored as identical matches by thesequence alignment program ALIGN-2 in that program's alignment of C andD, and where Z is the total number of nucleotides in D. It will beappreciated that where the length of nucleic acid sequence C is notequal to the length of nucleic acid sequence D, the % nucleic acidsequence identity of C to D will not equal the % nucleic acid sequenceidentity of D to C. As examples of % nucleic acid sequence identitycalculations, Tables 4 and 5, demonstrate how to calculate the % nucleicacid sequence identity of the nucleic acid sequence designated“Comparison DNA” to the nucleic acid sequence designated “PRO-DNA”,wherein “PRO-DNA” represents a hypothetical PRO-encoding nucleic acidsequence of interest, “Comparison DNA” represents the nucleotidesequence of a nucleic acid molecule against which the “PRO-DNA” nucleicacid molecule of interest is being compared, and “N”, “L” and “V” eachrepresent different hypothetical nucleotides. Unless specifically statedotherwise, all % nucleic acid sequence identity values used herein areobtained as described in the immediately preceding paragraph using theALIGN-2 computer program.

The invention also provides PRO226, PRO257, PRO268, PRO290, PRO36006,PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125, PRO1134,PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433,PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341,PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737,PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873,PRO10196, PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 orPRO85161 variant polynucleotides which are nucleic acid molecules thatencode a PRO226, PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365,PRO382, PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281,PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550,PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369,PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993,PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196,PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161polypeptide and which are capable of hybridizing, preferably understringent hybridization and wash conditions, to nucleotide sequencesencoding a full-length PRO226, PRO257, PRO268, PRO290, PRO36006, PRO363,PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155,PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474,PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348,PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800,PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196,PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161polypeptide as disclosed herein. PRO226, PRO257, PRO268, PRO290,PRO36006, PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125,PRO1134, PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419,PRO1433, PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193,PRO4341, PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989,PRO5737, PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852,PRO9873, PRO10196, PRO34778, PRO20233, PRO21956, PRO57290, PRO38465,PRO38683 or PRO85161 variant polypeptides may be those that are encodedby a PRO226, PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382,PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343,PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571,PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381,PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017,PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196, PRO34778,PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161 variantpolynucleotide.

The term “full-length coding region” when used in reference to a nucleicacid encoding a PRO226, PRO257, PRO268, PRO290, PRO36006, PRO363,PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155,PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474,PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348,PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800,PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196,PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161polypeptide refers to the sequence of nucleotides which encode thefull-length PRO226, PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365,PRO382, PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281,PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550,PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369,PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993,PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196,PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161polypeptide of the invention (which is often shown between start andstop codons, inclusive thereof, in the accompanying figures). The term“full-length coding region” when used in reference to an ATCC depositednucleic acid refers to the PRO226, PRO257, PRO268, PRO290, PRO36006,PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125, PRO1134,PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433,PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341,PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737,PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873,PRO10196, PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 orPRO85161 polypeptide-encoding portion of the cDNA that is inserted intothe vector deposited with the ATCC (which is often shown between startand stop codons, inclusive thereof, in the accompanying figures).

“Isolated,” when used to describe the various polypeptides disclosedherein, means polypeptide that has been identified and separated and/orrecovered from a component of its natural environment. Contaminantcomponents of its natural environment are materials that would typicallyinterfere with diagnostic or therapeutic uses for the polypeptide, andmay include enzymes, hormones, and other proteinaceous ornon-proteinaceous solutes. The invention provides that the polypeptidewill be purified (1) to a degree sufficient to obtain at least 15residues of N-terminal or internal amino acid sequence by use of aspinning cup sequenator, or (2) to homogeneity by SDS-PAGE undernon-reducing or reducing conditions using Coomassie blue or, preferably,silver stain. Isolated polypeptide includes polypeptide in situ withinrecombinant cells, since at least one component of the PRO226, PRO257,PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382, PRO444, PRO705,PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343, PRO1379, PRO1380,PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571, PRO1572, PRO1759,PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381, PRO4407, PRO4425,PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017, PRO7174, PRO9744,PRO9821, PRO9852, PRO9873, PRO10196, PRO34778, PRO20233, PRO21956,PRO57290, PRO38465, PRO38683 or PRO85161 polypeptide natural environmentwill not be present. Ordinarily, however, isolated polypeptide will beprepared by at least one purification step.

An “isolated” PRO226, PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365,PRO382, PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281,PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550,PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369,PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993,PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196,PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161polypeptide-encoding nucleic acid or other polypeptide-encoding nucleicacid is a nucleic acid molecule that is identified and separated from atleast one contaminant nucleic acid molecule with which it is ordinarilyassociated in the natural source of the polypeptide-encoding nucleicacid. An isolated polypeptide-encoding nucleic acid molecule is otherthan in the form or setting in which it is found in nature. Isolatedpolypeptide-encoding nucleic acid molecules therefore are distinguishedfrom the specific polypeptide-encoding nucleic acid molecule as itexists in natural cells. However, an isolated polypeptide-encodingnucleic acid molecule includes polypeptide-encoding nucleic acidmolecules contained in cells that ordinarily express the polypeptidewhere, for example, the nucleic acid molecule is in a chromosomallocation different from that of natural cells.

The term “control sequences” refers to DNA sequences necessary for theexpression of an operably linked coding sequence in a particular hostorganism. The control sequences that are suitable for prokaryotes, forexample, include a promoter, optionally an operator sequence, and aribosome binding site. Eukaryotic cells are known to utilize promoters,polyadenylation signals, and enhancers.

Nucleic acid is “operably linked” when it is placed into a functionalrelationship with another nucleic acid sequence. For example, DNA for apresequence or secretory leader is operably linked to DNA for apolypeptide if it is expressed as a preprotein that participates in thesecretion of the polypeptide; a promoter or enhancer is operably linkedto a coding sequence if it affects the transcription of the sequence; ora ribosome binding site is operably linked to a coding sequence if it ispositioned so as to facilitate translation. Generally, “operably linked”means that the DNA sequences being linked are contiguous, and, in thecase of a secretory leader, contiguous and in reading phase. However,enhancers do not have to be contiguous. Linking is accomplished byligation at convenient restriction sites. If such sites do not exist,the synthetic oligonucleotide adaptors or linkers are used in accordancewith conventional practice.

“Stringency” of hybridization reactions is readily determinable by oneof ordinary skill in the art, and generally is an empirical calculationdependent upon probe length, washing temperature, and saltconcentration. In general, longer probes require higher temperatures forproper annealing, while shorter probes need lower temperatures.Hybridization generally depends on the ability of denatured DNA toreanneal when complementary strands are present in an environment belowtheir melting temperature. The higher the degree of desired homologybetween the probe and hybridizable sequence, the higher the relativetemperature which can be used. As a result, it follows that higherrelative temperatures would tend to make the reaction conditions morestringent, while lower temperatures less so. For additional details andexplanation of stringency of hybridization reactions, see Ausubel etal., Current Protocols in Molecular Biology, Wiley IntersciencePublishers, (1995).

“Stringent conditions” or “high stringency conditions”, as definedherein, may be identified by those that: (1) employ low ionic strengthand high temperature for washing, for example 0.015 M sodiumchloride/0.0015 M sodium citrate/0.1% sodium dodecyl sulfate at 50° C.;(2) employ during hybridization a denaturing agent, such as formamide,for example, 50% (v/v) formamide with 0.1% bovine serum albumin/0.1%Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5with 750 mM sodium chloride, 75 mM sodium citrate at 42° C.; or (3)employ 50% formamide, 5×SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mMsodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5×Denhardt'ssolution, sonicated salmon sperm DNA (50 μg/ml), 0.1% SDS, and 10%dextran sulfate at 42° C., with washes at 42° C. in 0.2×SSC (sodiumchloride/sodium citrate) and 50% formamide at 55° C., followed by ahigh-stringency wash consisting of 0.1×SSC containing EDTA at 55° C.

“Moderately stringent conditions” may be identified as described bySambrook et al., Molecular Cloning: A Laboratory Manual, New York: ColdSpring Harbor Press, 1989, and include the use of washing solution andhybridization conditions (e.g., temperature, ionic strength and % SDS)less stringent that those described above. An example of moderatelystringent conditions is overnight incubation at 37° C. in a solutioncomprising: 20% formamide, 5×SSC (150 mM NaCl, 15 mM trisodium citrate),50 mM sodium phosphate (pH 7.6), 5×Denhardt's solution, 10% dextransulfate, and 20 mg/ml denatured sheared salmon sperm DNA, followed bywashing the filters in 1×SSC at about 37-50° C. The skilled artisan willrecognize how to adjust the temperature, ionic strength, etc. asnecessary to accommodate factors such as probe length and the like.

The term “epitope tagged” when used herein refers to a chimericpolypeptide comprising a PRO226, PRO257, PRO268, PRO290, PRO36006,PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125, PRO1134,PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433,PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341,PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737,PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873,PRO10196, PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 orPRO85161 polypeptide fused to a “tag polypeptide”. The tag polypeptidehas enough residues to provide an epitope against which an antibody canbe made, yet is short enough such that it does not interfere withactivity of the polypeptide to which it is fused. The tag polypeptidepreferably also is fairly unique so that the antibody does notsubstantially cross-react with other epitopes. Suitable tag polypeptidesgenerally have at least six amino acid residues and usually betweenabout 8 and 50 amino acid residues preferably, between about 10 and 20amino acid residues).

“Active” or “activity” for the purposes herein refers to form(s) of aPRO226, PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382,PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343,PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571,PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381,PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017,PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196, PRO34778,PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161 polypeptidewhich retain a biological and/or an immunological activity of native ornaturally-occurring PRO226, PRO257, PRO268, PRO290, PRO36006, PRO363,PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155,PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474,PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348,PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800,PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196,PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161polypeptide, wherein “biological” activity refers to a biologicalfunction (either inhibitory or stimulatory) caused by a native ornaturally-occurring PRO226, PRO257, PRO268, PRO290, PRO36006, PRO363,PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155,PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474,PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348,PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800,PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196,PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161polypeptide other than the ability to induce the production of anantibody against an antigenic epitope possessed by a native ornaturally-occurring PRO226, PRO257, PRO268, PRO290, PRO36006, PRO363,PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155,PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474,PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348,PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800,PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196,PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161polypeptide and an “immunological” activity refers to the ability toinduce the production of an antibody against an antigenic epitopepossessed by a native or naturally-occurring PRO226, PRO257, PRO268,PRO290, PRO36006, PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071,PRO1125, PRO1134, PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387,PRO1419, PRO1433, PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904,PRO35193, PRO4341, PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985,PRO4989, PRO5737, PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821,PRO9852, PRO9873, PRO10196, PRO34778, PRO20233, PRO21956, PRO57290,PRO38465, PRO38683 or PRO85161 polypeptide.

The term “antagonist” is used in the broadest sense [unless otherwisequalified], and includes any molecule that partially or fully blocks,inhibits, or neutralizes a biological activity of a native PRO226,PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382, PRO444,PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343, PRO1379,PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571, PRO1572,PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381, PRO4407,PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017, PRO7174,PRO9744, PRO9821, PRO9852, PRO9873, PRO10196, PRO34778, PRO20233,PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161 polypeptide disclosedherein. In a similar manner, the term “agonist” is used in the broadestsense [unless otherwise qualified] and includes any molecule that mimicsa biological activity of a native PRO226, PRO257, PRO268, PRO290,PRO36006, PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125,PRO1134, PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419,PRO1433, PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193,PRO4341, PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989,PRO5737, PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852,PRO9873, PRO10196, PRO34778, PRO20233, PRO21956, PRO57290, PRO38465,PRO38683 or PRO85161 polypeptide disclosed herein. Suitable agonist orantagonist molecules specifically include agonist or antagonistantibodies or antibody fragments, fragments or amino acid sequencevariants of native PRO226, PRO257, PRO268, PRO290, PRO36006, PRO363,PRO365, PRO382, PRO444, PRO705, PRO171, PRO1125, PRO1134, PRO1155,PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474,PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348,PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800,PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196,PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161polypeptides, peptides, antisense oligonucleotides, small organicmolecules, etc. Methods for identifying agonists or antagonists of aPRO226, PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382,PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343,PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571,PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381,PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017,PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196, PRO34778,PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161 polypeptidemay comprise contacting a PRO226, PRO257, PRO268, PRO290, PRO36006,PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125, PRO1134,PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433,PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341,PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737,PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873,PRO10196, PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 orPRO85161 polypeptide with a candidate agonist or antagonist molecule andmeasuring a detectable change in one or more biological activitiesnormally associated with the PRO226, PRO257, PRO268, PRO290, PRO36006,PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125, PRO1134,PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433,PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341,PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737,PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873,PRO10196, PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 orPRO85161 polypeptide.

“Treating” or “treatment” or “alleviation” refers to both therapeutictreatment and prophylactic or preventative measures, wherein the objectis to prevent or slow down (lessen) the targeted pathologic condition ordisorder. A subject in need of treatment may already have the disorder,or may be prone to have the disorder or may be in whom the disorder isto be prevented.

“Chronic” administration refers to administration of the agent(s) in acontinuous mode as opposed to an acute mode, so as to maintain theinitial therapeutic effect (activity) for an extended period of time.“Intermittent” administration is treatment that is not consecutivelydone without interruption, but rather is cyclic in nature.

“Mammal” for purposes of treatment refers to any animal classified as amammal, including humans, rodents such as rats or mice, domestic andfarm animals, and zoo, sports, or pet animals, such as dogs, cats,cattle, horses, sheep, pigs, goats, rabbits, etc. Preferably, the mammalis human.

Administration “in combination with” one or more further therapeuticagents includes simultaneous (concurrent) and consecutive administrationin any order.

“Carriers” as used herein include pharmaceutically acceptable carriers,excipients, or stabilizers which are nontoxic to the cell or mammalbeing exposed thereto at the dosages and concentrations employed. Oftenthe physiologically acceptable carrier is an aqueous pH bufferedsolution. Examples of physiologically acceptable carriers includebuffers such as phosphate, citrate, and other organic acids;antioxidants including ascorbic acid; low molecular weight (less thanabout 10 residues) polypeptide; proteins, such as serum albumin,gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, arginine or lysine; monosaccharides, disaccharides, andother carbohydrates including glucose, mannose, or dextrins; chelatingagents such as EDTA; sugar alcohols such as mannitol or sorbitol;salt-forming counterions such as sodium; and/or nonionic surfactantssuch as TWEEN™, polyethylene glycol (PEG), and PLURONICS™.

By “solid phase” is meant anon-aqueous matrix to which the antibody ofthe present invention can adhere. Examples of solid phases encompassedherein include those formed partially or entirely of glass (e.g.,controlled pore glass), polysaccharides (e.g., agarose),polyacrylamides, polystyrene, polyvinyl alcohol and silicones. Dependingon the context, the solid phase can comprise the well of an assay plate;in others it is a purification column (e.g., an affinity chromatographycolumn). This term also includes a discontinuous solid phase of discreteparticles, such as those described in U.S. Pat. No. 4,275,149.

A “liposome” is a small vesicle composed of various types of lipids,phospholipids and/or surfactant which is useful for delivery of a drug(such as a PRO226, PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365,PRO382, PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281,PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550,PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369,PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993,PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196,PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161polypeptide or antibody thereto) to a mammal. The components of theliposome are commonly arranged in a bilayer formation, similar to thelipid arrangement of biological membranes.

A “small molecule” is defined herein to have a molecular weight belowabout 500 Daltons.

An “effective amount” of a PRO226, PRO257, PRO268, PRO290, PRO36006,PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125, PRO1134,PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433,PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341,PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737,PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873,PRO10196, PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 orPRO85161 polypeptide, an anti-PRO226, anti-PRO257, anti-PRO268,anti-PRO290, anti-PRO36006, anti-PRO363, anti-PRO365, anti-PRO382,anti-PRO444, anti-PRO705, anti-PRO1071, anti-PRO1125, anti-PRO1134,anti-PRO1155, anti-PRO1281, anti-PRO1343, anti-PRO1379, anti-PRO1380,anti-PRO1387, anti-PRO1419, anti-PRO1433, anti-PRO1474, anti-PRO1550,anti-PRO1571, anti-PRO1572, anti-PRO1759, anti-PRO1904, anti-PRO35193,anti-PRO4341, anti-PRO4348, anti-PRO4369, anti-PRO4381, anti-PRO4407,anti-PRO4425, anti-PRO4985, anti-PRO4989, anti-PRO5737, anti-PRO5800,anti-PRO5993, anti-PRO6017, anti-PRO7174, anti-PRO9744, anti-PRO9821,anti-PRO9852, anti-PRO9873, anti-PRO10196, anti-PRO34778, anti-PRO20233,anti-PRO21956, anti-PRO57290, anti-PRO38465, anti-PRO38683 oranti-PRO85161 antibody, a PRO226, PRO257, PRO268, PRO290, PRO36006,PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125, PRO1134,PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433,PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341,PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737,PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873,PRO10196, PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 orPRO85161 binding oligopeptide, a PRO226, PRO257, PRO268, PRO290,PRO36006, PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125,PRO1134, PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419,PRO1433, PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193,PRO4341, PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989,PRO5737, PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852,PRO9873, PRO10196, PRO34778, PRO20233, PRO21956, PRO57290, PRO38465,PRO38683 or PRO85161 binding organic molecule or an agonist orantagonist thereof as disclosed herein is an amount sufficient to carryout a specifically stated purpose. An “effective amount” may bedetermined empirically and in a routine manner, in relation to thestated purpose.

The term “therapeutically effective amount” refers to an amount of ananti-PRO226, anti-PRO257, anti-PRO268, anti-PRO290, anti-PRO36006,anti-PRO363, anti-PRO365, anti-PRO382, anti-PRO444, anti-PRO705,anti-PRO1071, anti-PRO1125, anti-PRO1134, anti-PRO1155, anti-PRO1281,anti-PRO1343, anti-PRO1379, anti-PRO1380, anti-PRO1387, anti-PRO1419,anti-PRO1433, anti-PRO1474, anti-PRO1550, anti-PRO1571, anti-PRO1572,anti-PRO1759, anti-PRO1904, anti-PRO35193, anti-PRO4341, anti-PRO4348,anti-PRO4369, anti-PRO4381, anti-PRO4407, anti-PRO4425, anti-PRO4985,anti-PRO4989, anti-PRO5737, anti-PRO5800, anti-PRO5993, anti-PRO6017,anti-PRO7174, anti-PRO9744, anti-PRO9821, anti-PRO9852, anti-PRO9873,anti-PRO10196, anti-PRO34778, anti-PRO20233, anti-PRO21956,anti-PRO57290, anti-PRO38465, anti-PRO38683 or anti-PRO85161 antibody, aPRO226, PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382,PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343,PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571,PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381,PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017,PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196, PRO34778,PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161polypeptide, a PRO226, PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365,PRO382, PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281,PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550,PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369,PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993,PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196,PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161binding oligopeptide, a PRO226, PRO257, PRO268, PRO290, PRO36006,PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125, PRO1134,PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433,PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341,PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737,PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873,PRO10196, PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 orPRO85161 binding organic molecule or other drug effective to “treat” adisease or disorder in a subject or mammal. In the case of cancer, thetherapeutically effective amount of the drug may reduce the number ofcancer cells; reduce the tumor size; inhibit (i.e., slow to some extentand preferably stop) cancer cell infiltration into peripheral organs;inhibit (i.e., slow to some extent and preferably stop) tumormetastasis; inhibit, to some extent, tumor growth; and/or relieve tosome extent one or more of the symptoms associated with the cancer. Seethe definition herein of “treating”. To the extent the drug may preventgrowth and/or kill existing cancer cells, it may be cytostatic and/orcytotoxic.

The phrases “cardiovascular, endothelial and angiogenic disorder”,“cardiovascular, endothelial and angiogenic dysfunction”,“cardiovascular, endothelial or angiogenic disorder” and“cardiovascular, endothelial or angiogenic dysfunction” are usedinterchangeably and refer in part to systemic disorders that affectvessels, such as diabetes mellitus, as well as diseases of the vesselsthemselves, such as of the arteries, capillaries, veins, and/orlymphatics. This would include indications that stimulate angiogenesisand/or cardiovascularization, and those that inhibit angiogenesis and/orcardiovascularization. Such disorders include, for example, arterialdisease, such as atherosclerosis, hypertension, inflammatoryvasculitides, Reynaud's disease and Reynaud's phenomenon, aneurysms, andarterial restenosis; venous and lymphatic disorders such asthrombophlebitis, lymphangitis, and lymphedema; and other vasculardisorders such as peripheral vascular disease, cancer such as vasculartumors, e.g., hemangioma (capillary and cavernous), glomus tumors,telangiectasia, bacillary angiomatosis, hemangioendothelioma,angiosarcoma, haemangiopericytoma, Kaposi's sarcoma, lymphangioma, andlymphangiosarcoma, tumor angiogenesis, trauma such as wounds, burns, andother injured tissue, implant fixation, scarring, ischemia reperfusioninjury, rheumatoid arthritis, cerebrovascular disease, renal diseasessuch as acute renal failure, or osteoporosis. This would also includeangina, myocardial infarctions such as acute myocardial infarctions,cardiac hypertrophy, and heart failure such as CHF.

“Hypertrophy”, as used herein, is defined as an increase in mass of anorgan or structure independent of natural growth that does not involvetumor formation. Hypertrophy of an organ or tissue is due either to anincrease in the mass of the individual cells (true hypertrophy), or toan increase in the number of cells making up the tissue (hyperplasia),or both. Certain organs, such as the heart, lose the ability to divideshortly after birth. Accordingly, “cardiac hypertrophy” is defined as anincrease in mass of the heart, which, in adults, is characterized by anincrease in myocyte cell size and contractile protein content withoutconcomitant cell division. The character of the stress responsible forinciting the hypertrophy, (e.g., increased preload, increased afterload,loss of myocytes, as in myocardial infarction, or primary depression ofcontractility), appears to play a critical role in determining thenature of the response. The early stage of cardiac hypertrophy isusually characterized morphologically by increases in the size ofmyofibrils and mitochondria, as well as by enlargement of mitochondriaand nuclei. At this stage, while muscle cells are larger than normal,cellular organization is largely preserved. At a more advanced stage ofcardiac hypertrophy, there are preferential increases in the size ornumber of specific organelles, such as mitochondria, and new contractileelements are added in localized areas of the cells, in an irregularmanner. Cells subjected to long-standing hypertrophy show more obviousdisruptions in cellular organization, including markedly enlarged nucleiwith highly lobulated membranes, which displace adjacent myofibrils andcause breakdown of normal Z-band registration. The phrase “cardiachypertrophy” is used to include all stages of the progression of thiscondition, characterized by various degrees of structural damage of theheart muscle, regardless of the underlying cardiac disorder. Hence, theterm also includes physiological conditions instrumental in thedevelopment of cardiac hypertrophy, such as elevated blood pressure,aortic stenosis, or myocardial infarction.

“Heart failure” refers to an abnormality of cardiac function where theheart does not pump blood at the rate needed for the requirements ofmetabolizing tissues. The heart failure can be caused by a number offactors, including ischemic, congenital, rheumatic, or idiopathic forms.

“Congestive heart failure” (CHF) is a progressive pathologic state wherethe heart is increasingly unable to supply adequate cardiac output (thevolume of blood pumped by the heart over time) to deliver the oxygenatedblood to peripheral tissues. As CHF progresses, structural andhemodynamic damages occur. While these damages have a variety ofmanifestations, one characteristic symptom is ventricular hypertrophy.CHF is a common end result of a number of various cardiac disorders.

“Myocardial infarction” generally results from atherosclerosis of thecoronary arteries, often with superimposed coronary thrombosis. It maybe divided into two major types: transmural infarcts, in whichmyocardial necrosis involves the full thickness of the ventricular wall,and subendocardial (nontransmural) infarcts, in which the necrosisinvolves the subendocardium, the intramural myocardium, or both, withoutextending all the way through the ventricular wall to the epicardium.Myocardial infarction is known to cause both a change in hemodynamiceffects and an alteration in structure in the damaged and healthy zonesof the heart. Thus, for example, myocardial infarction reduces themaximum cardiac output and the stroke volume of the heart. Alsoassociated with myocardial infarction is a stimulation of the DNAsynthesis occurring in the interstice as well as an increase in theformation of collagen in the areas of the heart not affected.

As a result of the increased stress or strain placed on the heart inprolonged hypertension due, for example, to the increased totalperipheral resistance, cardiac hypertrophy has long been associated with“hypertension”. A characteristic of the ventricle that becomeshypertrophic as a result of chronic pressure overload is an impaireddiastolic performance. Fouad et al., J. Am. Coll. Cardiol., 4: 1500-1506(1984); Smith et al., J. Am. Coll. Cardiol., 5: 869-874 (1985). Aprolonged left ventricular relaxation has been detected in earlyessential hypertension, in spite of normal or supranormal systolicfunction. Hartford et al., Hypertension, 6: 329-338 (1984). However,there is no close parallelism between blood pressure levels and cardiachypertrophy. Although improvement in left ventricular function inresponse to antihypertensive therapy has been reported in humans,patients variously treated with a diuretic (hydrochlorothiazide), aβ-blocker (propranolol), or a calcium channel blocker (diltiazem), haveshown reversal of left ventricular hypertrophy, without improvement indiastolic function. Inouye et al., Am. J. Cardiol., 53: 1583-7 (1984).

Another complex cardiac disease associated with cardiac hypertrophy is“hypertrophic cardiomyopathy”. This condition is characterized by agreat diversity of morphologic, functional, and clinical features (Maronet al., N. Engl. J. Med., 316: 780-789 (1987); Spirito et al., N. Engl.J. Med., 320: 749-755 (1989); Louie and Edwards, Prog. Cardiovasc. Dis.,36: 275-308 (1994); Wigle et al., Circulation, 92: 1680-1692 (1995)),the heterogeneity of which is accentuated by the fact that it afflictspatients of all ages. Spirito et al., N. Engl. J. Med., 336: 775-785(1997). The causative factors of hypertrophic cardiomyopathy are alsodiverse and little understood. In general, mutations in genes encodingsarcomeric proteins are associated with hypertrophic cardiomyopathy.Recent data suggest that β-myosin heavy chain mutations may account forapproximately 30 to 40 percent of cases of familial hypertrophiccardiomyopathy. Watkins et al., N. Engl. J. Med., 326: 1108-1114 (1992);Schwartz et al., Circulation, 91: 532-540 (1995); Marian and Roberts,Circulation, 92: 1336-1347 (1995); Thierfelder et al., Cell, 77: 701-712(1994); Watkins et al., Nat. Gen., 11: 434-437 (1995). Besides β-myosinheavy chain, other locations of genetic mutations include cardiactroponin T, alpha topomyosin, cardiac myosin binding protein C,essential myosin light chain, and regulatory myosin light chain. See,Malik and Watkins, Curr. Opin. Cardiol., 12: 295-302 (1997).

Supravalvular “aortic stenosis” is an inherited vascular disordercharacterized by narrowing of the ascending aorta, but other arteries,including the pulmonary arteries, may also be affected. Untreated aorticstenosis may lead to increased intracardiac pressure resulting inmyocardial hypertrophy and eventually heart failure and death. Thepathogenesis of this disorder is not fully understood, but hypertrophyand possibly hyperplasia of medial smooth muscle are prominent featuresof this disorder. It has been reported that molecular variants of theelastin gene are involved in the development and pathogenesis of aorticstenosis. U.S. Pat. No. 5,650,282 issued Jul. 22, 1997.

“Valvular regurgitation” occurs as a result of heart diseases resultingin disorders of the cardiac valves. Various diseases, like rheumaticfever, can cause the shrinking or pulling apart of the valve orifice,while other diseases may result in endocarditis, an inflammation of theendocardium or lining membrane of the atrioventricular orifices andoperation of the heart. Defects such as the narrowing of the valvestenosis or the defective closing of the valve result in an accumulationof blood in the heart cavity or regurgitation of blood past the valve.If uncorrected, prolonged valvular stenosis or insufficiency may resultin cardiac hypertrophy and associated damage to the heart muscle, whichmay eventually necessitate valve replacement.

The term “immune related disease” means a disease in which a componentof the immune system of a mammal causes, mediates or otherwisecontributes to a morbidity in the mammal. Also included are diseases inwhich stimulation or intervention of the immune response has anameliorative effect on progression of the disease. Included within thisterm are immune-mediated inflammatory diseases, non-immune-mediatedinflammatory diseases, infectious diseases, immunodeficiency diseases,neoplasia, etc.

The term “T cell mediated disease” means a disease in which T cellsdirectly or indirectly mediate or otherwise contribute to a morbidity ina mammal. The T cell mediated disease may be associated with cellmediated effects, lymphokine mediated effects, etc., and even effectsassociated with B cells if the B cells are stimulated, for example, bythe lymphokines secreted by T cells.

Examples of immune-related and inflammatory diseases, some of which areimmune or T cell mediated, include systemic lupus erythematosis,rheumatoid arthritis, juvenile chronic arthritis, spondyloarthropathies,systemic sclerosis (scleroderma), idiopathic inflammatory myopathies(dermatomyositis, polymyositis), Sjögren's syndrome, systemicvasculitis, sarcoidosis, autoimmune hemolytic anemia (immunepancytopenia, paroxysmal nocturnal hemoglobinuria), autoimmunethrombocytopenia (idiopathic thrombocytopenic purpura, immune-mediatedthrombocytopenia), thyroiditis (Grave's disease, Hashimoto'sthyroiditis, juvenile lymphocytic thyroiditis, atrophic thyroiditis),diabetes mellitus, immune-mediated renal disease (glomerulonephritis,tubulointerstitial nephritis), demyelinating diseases of the central andperipheral nervous systems such as multiple sclerosis, idiopathicdemyelinating polyneuropathy or Guillain-Barré syndrome, and chronicinflammatory demyelinating polyneuropathy, hepatobiliary diseases suchas infectious hepatitis (hepatitis A, B, C, D, E and othernon-hepatotropic viruses), autoimmune chronic active hepatitis, primarybiliary cirrhosis, granulomatous hepatitis, and sclerosing cholangitis,inflammatory bowel disease (ulcerative colitis: Crohn's disease),gluten-sensitive enteropathy, and Whipple's disease, autoimmune orimmune-mediated skin diseases including bullous skin diseases, erythemamultiforme and contact dermatitis, psoriasis, allergic diseases such asasthma, allergic rhinitis, atopic dermatitis, food hypersensitivity andurticaria, immunologic diseases of the lung such as eosinophilicpneumonia, idiopathic pulmonary fibrosis and hypersensitivitypneumonitis, or transplantation associated diseases including graftrejection and graft-versus-host-disease. Infectious diseases includingviral diseases such as AIDS (HIV infection), hepatitis A, B, C, D, andE, herpes, etc., bacterial infections, fungal infections, protozoalinfections and parasitic infections.

An “autoimmune disease” herein is a disease or disorder arising from anddirected against an individual's own tissues or a co-segregate ormanifestation thereof or resulting condition therefrom. Examples ofautoimmune diseases or disorders include, but are not limited toarthritis (rheumatoid arthritis, juvenile rheumatoid arthritis,osteoarthritis, psoriatic arthritis, and ankylosing spondylitis),psoriasis, dermatitis including atopic dermatitis; chronic idiopathicurticaria, including chronic autoimmune urticaria,polymyositis/dermatomyositis, toxic epidermal necrolysis, systemicscleroderma and sclerosis, responses associated with inflammatory boweldisease (IBD) (Crohn's disease, ulcerative colitis), and IBD withco-segregate of pyoderma gangrenosum, erythema nodosum, primarysclerosing cholangitis, and/or episcleritis), respiratory distresssyndrome, including adult respiratory distress syndrome (ARDS),meningitis, IgE-mediated diseases such as anaphylaxis and allergicrhinitis, encephalitis such as Rasmussen's encephalitis, uveitis,colitis such as microscopic colitis and collagenous colitis,glomerulonephritis (GN) such as membranous GN, idiopathic membranous GN,membranous proliferative GN (MPGN), including Type I and Type II, andrapidly progressive GN, allergic conditions, eczema, asthma, conditionsinvolving infiltration of T cells and chronic inflammatory responses,atherosclerosis, autoimmune myocarditis, leukocyte adhesion deficiency,systemic lupus erythematosus (SLE) such as cutaneous SLE, lupus(including nephritis, cerebritis, pediatric, non-renal, discoid,alopecia), juvenile onset diabetes, multiple sclerosis (MS) such asspino-optical MS, allergic encephalomyelitis, immune responsesassociated with acute and delayed hypersensitivity mediated by cytokinesand T-lymphocytes, tuberculosis, sarcoidosis, granulomatosis includingWegener's granulomatosis, agranulocytosis, vasculitis (including LargeVessel vasculitis (including Polymyalgia Rheumatica and Giant Cell(Takayasu's) Arteritis), Medium Vessel vasculitis (including Kawasaki'sDisease and Polyarteritis Nodosa), CNS vasculitis, and ANCA-associatedvasculitis, such as Churg-Strauss vasculitis or syndrome (CSS)),aplastic anemia, Coombs positive anemia, Diamond Blackfan anemia, immunehemolytic anemia including autoimmune hemolytic anemia (AIHA),pernicious anemia, pure red cell aplasia (PRCA), Factor VIII deficiency,hemophilia A, autoimmune neutropenia, pancytopenia, leukopenia, diseasesinvolving leukocyte diapedesis, CNS inflammatory disorders, multipleorgan injury syndrome, myasthenia gravis, antigen-antibody complexmediated diseases, anti-glomerular basement membrane disease,anti-phospholipid antibody syndrome, allergic neuritis, Bechet disease,Castleman's syndrome, Goodpasture's Syndrome, Lambert-Eaton MyasthenicSyndrome, Reynaud's syndrome, Sjorgen's syndrome, Stevens-Johnsonsyndrome, solid organ transplant rejection (including pretreatment forhigh panel reactive antibody titers, IgA deposit in tissues, andrejection arising from renal transplantation, liver transplantation,intestinal transplantation, cardiac transplantation, etc.), graft versushost disease (GVHD), pemphigoid bullous, pemphigus (including vulgaris,foliaceus, and pemphigus mucus-membrane pemphigoid), autoimmunepolyendocrinopathies, Reiter's disease, stiff-man syndrome, immunecomplex nephritis, IgM polyneuropathies or IgM mediated neuropathy,idiopathic thrombocytopenic purpura (ITP), thrombotic throbocytopenicpurpura (TTP), thrombocytopenia (as developed by myocardial infarctionpatients, for example), including autoimmune thrombocytopenia,autoimmune disease of the testis and ovary including autoimmune orchitisand oophoritis, primary hypothyroidism; autoimmune endocrine diseasesincluding autoimmune thyroiditis, chronic thyroiditis (Hashimoto'sThyroiditis), subacute thyroiditis, idiopathic hypothyroidism, Addison'sdisease, Grave's disease, autoimmune polyglandular syndromes (orpolyglandular endocrinopathy syndromes), Type I diabetes also referredto as insulin-dependent diabetes mellitus (IDDM), including pediatricIDDM, and Sheehan's syndrome; autoimmune hepatitis, Lymphoidinterstitial pneumonitis (HIV), bronchiolitis obliterans(non-transplant) vs NSIP, Guillain-Barré Syndrome, Berger's Disease (IgAnephropathy), primary biliary cirrhosis, celiac sprue (glutenenteropathy), refractory sprue with co-segregate dermatitisherpetiformis, cryoglobulinemia, amylotrophic lateral sclerosis (ALS;Lou Gehrig's disease), coronary artery disease, autoimmune inner eardisease (AIED), autoimmune hearing loss, opsoclonus myoclonus syndrome(OMS), polychondritis such as refractory polychondritis, pulmonaryalveolar proteinosis, amyloidosis, giant cell hepatitis, scleritis,monoclonal gammopathy of uncertain/unknown significance (MGUS),peripheral neuropathy, paraneoplastic syndrome, channelopathies such asepilepsy, migraine, arrhythmia, muscular disorders, deafness, blindness,periodic paralysis, and channelopathies of the CNS; autism, inflammatorymyopathy, and focal segmental glomerulosclerosis (FSGS).

The phrase “anxiety related disorders” refers to disorders of anxiety,mood, and substance abuse, including but not limited to: depression,generalized anxiety disorders, attention deficit disorder, sleepdisorder, hyperactivity disorder, obsessive compulsive disorder,schizophrenia, cognitive disorders, hyperalgesia and sensory disorders.Such disorders include the mild to moderate anxiety, anxiety disorderdue to a general medical condition, anxiety disorder not otherwisespecified, generalized anxiety disorder, panic attack, panic disorderwith agoraphobia, panic disorder without agoraphobia, posttraumaticstress disorder, social phobia, social anxiety, autism, specific phobia,substance-induced anxiety disorder, acute alcohol withdrawal, obsessivecompulsive disorder, agoraphobia, monopolar disorders, bipolar disorderI or II, bipolar disorder not otherwise specified, cyclothymic disorder,depressive disorder, major depressive disorder, mood disorder,substance-induced mood disorder, enhancement of cognitive function, lossof cognitive function associated with but not limited to Alzheimer'sdisease, stroke, or traumatic injury to the brain, seizures resultingfrom disease or injury including but not limited to epilepsy, learningdisorders/disabilities, cerebral palsy. In addition, anxiety disordersmay apply to personality disorders including but not limited to thefollowing types: paranoid, antisocial, avoidant behavior, borderlinepersonality disorders, dependent, histrionic, narcissistic,obsessive-compulsive, schizoid, and schizotypal.

The term “lipid metabolic disorder” refers to abnormal clinicalchemistry levels of cholesterol and triglycerides, wherein elevatedlevels of these lipids is an indication for atherosclerosis.Additionally, abnormal serum lipid levels may be an indication ofvarious cardiovascular diseases including hypertension, stroke, coronaryartery diseases, diabetes and/or obesity.

The phrase “eye abnormality” refers to such potential disorders of theeye as they may be related to atherosclerosis or various opthalmologicalabnormalities. Such disorders include but are not limited to thefollowing: retinal dysplasia, various retinopathies, restenosis, retinalartery obstruction or occlusion; retinal degeneration causing secondaryatrophy of the retinal vasculature, retinitis pigmentosa, maculardystrophies, Stargardt's disease, congenital stationary night blindness,choroideremia, gyrate atrophy, Leber's congenital amaurosis,retinoschisis disorders, Wagner's syndrome, Usher syndromes, Zellwegersyndrome, Saldino-Mainzer syndrome, Senior-Loken syndrome, Bardet-Biedlsyndrome, Alport's syndrome, Alstrom's syndrome, Cockayne's syndrome,dysplaisa spondyloepiphysaria congentia, Flynn-Aird syndrome, Friedreichataxia, Hallgren syndrome, Marshall syndrome, Albers-Schnoberg disease,Refsum's disease, Kearns-Sayre syndrome, Waardenburg's syndrome, Alagilesyndrome, myotonic dystrophy, olivopontocerebellar atrophy, Pierre-Mariedunsdrome, Stickler syndrome, carotinemeia, cystinosis, Wolframsyndrome, Bassen-Kornzweig syndrome, abetalipoproteinemia, incontinentiapigmenti, Batten's disease, mucopolysaccharidoses, homocystinuria, ormannosidosis. Cataracts are also considered an eye abnormality and areassociated with such systemic diseases as: Human Down's syndrome,Hallerman-Streiff syndrome, Lowe syndrome, galactosemia, Marfansyndrome, Trismoy 13-15 condition, Alport syndrome, myotonic dystrophy,Fabry disease, hypothroidisms, or Conradi syndrome. Other oculardevelopmental anomalies include: Aniridia, anterior segment anddysgenesis syndrome. Cataracts may also occur as a result of anintraocular infection or inflammation (uveitis).

A “growth inhibitory amount” of an anti-PRO226, anti-PRO257,anti-PRO268, anti-PRO290, anti-PRO36006, anti-PRO363, anti-PRO365,anti-PRO382, anti-PRO444, anti-PRO705, anti-PRO1071, anti-PRO1125,anti-PRO1134, anti-PRO1155, anti-PRO1281, anti-PRO1343, anti-PRO1379,anti-PRO1380, anti-PRO1387, anti-PRO1419, anti-PRO1433, anti-PRO1474,anti-PRO1550, anti-PRO1571, anti-PRO1572, anti-PRO1759, anti-PRO1904,anti-PRO35193, anti-PRO4341, anti-PRO4348, anti-PRO4369, anti-PRO4381,anti-PRO4407, anti-PRO4425, anti-PRO4985, anti-PRO4989, anti-PRO5737,anti-PRO5800, anti-PRO5993, anti-PRO6017, anti-PRO7174, anti-PRO9744,anti-PRO9821, anti-PRO9852, anti-PRO9873, anti-PRO10196, anti-PRO34778,anti-PRO20233, anti-PRO21956, anti-PRO57290, anti-PRO38465,anti-PRO38683 or anti-PRO85161 antibody, PRO226, PRO257, PRO268, PRO290,PRO36006, PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125,PRO1134, PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419,PRO1433, PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193,PRO4341, PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989,PRO5737, PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852,PRO9873, PRO10196, PRO34778, PRO20233, PRO21956, PRO57290, PRO38465,PRO38683 or PRO85161 polypeptide, PRO226, PRO257, PRO268, PRO290,PRO36006, PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125,PRO1134, PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419,PRO1433, PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193,PRO4341, PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989,PRO5737, PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852,PRO9873, PRO10196, PRO34778, PRO20233, PRO21956, PRO57290, PRO38465,PRO38683 or PRO85161 binding oligopeptide or PRO226, PRO257, PRO268,PRO290, PRO36006, PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071,PRO1125, PRO1134, PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387,PRO1419, PRO1433, PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904,PRO35193, PRO4341, PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985,PRO4989, PRO5737, PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821,PRO9852, PRO9873, PRO10196, PRO34778, PRO20233, PRO21956, PRO57290,PRO38465, PRO38683 or PRO85161 binding organic molecule is an amountcapable of inhibiting the growth of a cell, especially tumor, e.g.,cancer cell, either in vitro or in vivo. A “growth inhibitory amount” ofan anti-PRO226, anti-PRO257, anti-PRO268, anti-PRO290, anti-PRO36006,anti-PRO363, anti-PRO365, anti-PRO382, anti-PRO444, anti-PRO705,anti-PRO1071, anti-PRO1125, anti-PRO1134, anti-PRO1155, anti-PRO1281,anti-PRO1343, anti-PRO1379, anti-PRO1380, anti-PRO1387, anti-PRO1419,anti-PRO1433, anti-PRO1474, anti-PRO1550, anti-PRO1571, anti-PRO1572,anti-PRO1759, anti-PRO1904, anti-PRO35193, anti-PRO4341, anti-PRO4348,anti-PRO4369, anti-PRO4381, anti-PRO4407, anti-PRO4425, anti-PRO4985,anti-PRO4989, anti-PRO5737, anti-PRO5800, anti-PRO5993, anti-PRO6017,anti-PRO7174, anti-PRO9744, anti-PRO9821, anti-PRO9852, anti-PRO9873,anti-PRO10196, anti-PRO34778, anti-PRO20233, anti-PRO21956,anti-PRO57290, anti-PRO38465, anti-PRO38683 or anti-PRO85161 antibody,PRO226, PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382,PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343,PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571,PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381,PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017,PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196, PRO34778,PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161polypeptide, PRO226, PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365,PRO382, PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281,PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550,PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369,PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993,PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196,PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161binding oligopeptide or PRO226, PRO257, PRO268, PRO290, PRO36006,PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125, PRO1134,PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433,PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341,PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737,PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873,PRO10196, PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 orPRO85161 binding organic molecule for purposes of inhibiting neoplasticcell growth may be determined empirically and in a routine manner.

A “cytotoxic amount” of an anti-PRO226, anti-PRO257, anti-PRO268,anti-PRO290, anti-PRO36006, anti-PRO363, anti-PRO365, anti-PRO382,anti-PRO444, anti-PRO705, anti-PRO1071, anti-PRO1125, anti-PRO1134,anti-PRO1155, anti-PRO1281, anti-PRO1343, anti-PRO1379, anti-PRO1380,anti-PRO1387, anti-PRO1419, anti-PRO1433, anti-PRO1474, anti-PRO1550,anti-PRO1571, anti-PRO1572, anti-PRO1759, anti-PRO1904, anti-PRO35193,anti-PRO4341, anti-PRO4348, anti-PRO4369, anti-PRO4381, anti-PRO4407,anti-PRO4425, anti-PRO4985, anti-PRO4989, anti-PRO5737, anti-PRO5800,anti-PRO5993, anti-PRO6017, anti-PRO7174, anti-PRO9744, anti-PRO9821,anti-PRO9852, anti-PRO9873, anti-PRO10196, anti-PRO34778, anti-PRO20233,anti-PRO21956, anti-PRO57290, anti-PRO38465, anti-PRO38683 oranti-PRO85161 antibody, PRO226, PRO257, PRO268, PRO290, PRO36006,PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125, PRO1134,PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433,PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341,PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737,PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873,PRO10196, PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 orPRO85161 polypeptide, PRO226, PRO257, PRO268, PRO290, PRO36006, PRO363,PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155,PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474,PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348,PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800,PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196,PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161binding oligopeptide or PRO226, PRO257, PRO268, PRO290, PRO36006,PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125, PRO1134,PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433,PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341,PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737,PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873,PRO10196, PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 orPRO85161 binding organic molecule is an amount capable of causing thedestruction of a cell, especially tumor, e.g., cancer cell, either invitro or in vivo. A “cytotoxic amount” of an anti-PRO226, anti-PRO257,anti-PRO268, anti-PRO290, anti-PRO36006, anti-PRO363, anti-PRO365,anti-PRO382, anti-PRO444, anti-PRO705, anti-PRO1071, anti-PRO1125,anti-PRO1134, anti-PRO1155, anti-PRO1281, anti-PRO1343, anti-PRO1379,anti-PRO1380, anti-PRO1387, anti-PRO1419, anti-PRO1433, anti-PRO1474,anti-PRO1550, anti-PRO1571, anti-PRO1572, anti-PRO1759, anti-PRO1904,anti-PRO35193, anti-PRO4341, anti-PRO4348, anti-PRO4369, anti-PRO4381,anti-PRO4407, anti-PRO4425, anti-PRO4985, anti-PRO4989, anti-PRO5737,anti-PRO5800, anti-PRO5993, anti-PRO6017, anti-PRO7174, anti-PRO9744,anti-PRO9821, anti-PRO9852, anti-PRO9873, anti-PRO10196, anti-PRO34778,anti-PRO20233, anti-PRO21956, anti-PRO57290, anti-PRO38465,anti-PRO38683 or anti-PRO85161 antibody, PRO226, PRO257, PRO268, PRO290,PRO36006, PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125,PRO1134, PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419,PRO1433, PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193,PRO4341, PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989,PRO5737, PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852,PRO9873, PRO10196, PRO34778, PRO20233, PRO21956, PRO57290, PRO38465,PRO38683 or PRO85161 polypeptide, PRO226, PRO257, PRO268, PRO290,PRO36006, PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125,PRO1134, PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419,PRO1433, PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193,PRO4341, PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989,PRO5737, PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852,PRO9873, PRO10196, PRO34778, PRO20233, PRO21956, PRO57290, PRO38465,PRO38683 or PRO85161 binding oligopeptide or PRO226, PRO257, PRO268,PRO290, PRO36006, PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071,PRO1125, PRO1134, PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387,PRO1419, PRO1433, PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904,PRO35193, PRO4341, PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985,PRO4989, PRO5737, PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821,PRO9852, PRO9873, PRO10196, PRO34778, PRO20233, PRO21956, PRO57290,PRO38465, PRO38683 or PRO85161 binding organic molecule for purposes ofinhibiting neoplastic cell growth may be determined empirically and in aroutine manner.

The term “antibody” is used in the broadest sense and specificallycovers, for example, single anti-PRO226, anti-PRO257, anti-PRO268,anti-PRO290, anti-PRO36006, anti-PRO363, anti-PRO365, anti-PRO382,anti-PRO444, anti-PRO705, anti-PRO1071, anti-PRO1125, anti-PRO1134,anti-PRO1155, anti-PRO1281, anti-PRO1343, anti-PRO1379, anti-PRO1380,anti-PRO1387, anti-PRO1419, anti-PRO1433, anti-PRO1474, anti-PRO1550,anti-PRO1571, anti-PRO1572, anti-PRO1759, anti-PRO1904, anti-PRO35193,anti-PRO4341, anti-PRO4348, anti-PRO4369, anti-PRO4381, anti-PRO4407,anti-PRO4425, anti-PRO4985, anti-PRO4989, anti-PRO5737, anti-PRO5800,anti-PRO5993, anti-PRO6017, anti-PRO7174, anti-PRO9744, anti-PRO9821,anti-PRO9852, anti-PRO9873, anti-PRO10196, anti-PRO34778, anti-PRO20233,anti-PRO21956, anti-PRO57290, anti-PRO38465, anti-PRO38683 oranti-PRO85161 antibody monoclonal antibodies (including agonist,antagonist, and neutralizing antibodies), anti-PRO226, anti-PRO257,anti-PRO268, anti-PRO290, anti-PRO36006, anti-PRO363, anti-PRO365,anti-PRO382, anti-PRO444, anti-PRO705, anti-PRO1071, anti-PRO1125,anti-PRO1134, anti-PRO1155, anti-PRO1281, anti-PRO1343, anti-PRO1379,anti-PRO1380, anti-PRO1387, anti-PRO1419, anti-PRO1433, anti-PRO1474,anti-PRO1550, anti-PRO1571, anti-PRO1572, anti-PRO1759, anti-PRO1904,anti-PRO35193, anti-PRO4341, anti-PRO4348, anti-PRO4369, anti-PRO4381,anti-PRO4407, anti-PRO4425, anti-PRO4985, anti-PRO4989, anti-PRO5737,anti-PRO5800, anti-PRO5993, anti-PRO6017, anti-PRO7174, anti-PRO9744,anti-PRO9821, anti-PRO9852, anti-PRO9873, anti-PRO10196, anti-PRO34778,anti-PRO20233, anti-PRO21956, anti-PRO57290, anti-PRO38465,anti-PRO38683 or anti-PRO85161 antibody compositions with polyepitopicspecificity, polyclonal antibodies, single chain anti-PRO226,anti-PRO257, anti-PRO268, anti-PRO290, anti-PRO36006, anti-PRO363,anti-PRO365, anti-PRO382, anti-PRO444, anti-PRO705, anti-PRO1071,anti-PRO1125, anti-PRO1134, anti-PRO1155, anti-PRO1281, anti-PRO1343,anti-PRO1379, anti-PRO1380, anti-PRO1387, anti-PRO1419, anti-PRO1433,anti-PRO1474, anti-PRO1550, anti-PRO1571, anti-PRO1572, anti-PRO1759,anti-PRO1904, anti-PRO35193, anti-PRO4341, anti-PRO4348, anti-PRO4369,anti-PRO4381, anti-PRO4407, anti-PRO4425, anti-PRO4985, anti-PRO4989,anti-PRO5737, anti-PRO5800, anti-PRO5993, anti-PRO6017, anti-PRO7174,anti-PRO9744, anti-PRO9821, anti-PRO9852, anti-PRO9873, anti-PRO10196,anti-PRO34778, anti-PRO20233, anti-PRO21956, anti-PRO57290,anti-PRO38465, anti-PRO38683 or anti-PRO85161 antibodies, and fragmentsof anti-PRO226, anti-PRO257, anti-PRO268, anti-PRO290, anti-PRO36006,anti-PRO363, anti-PRO365, anti-PRO382, anti-PRO444, anti-PRO705,anti-PRO1071, anti-PRO1125, anti-PRO1134, anti-PRO1155, anti-PRO1281,anti-PRO1343, anti-PRO1379, anti-PRO1380, anti-PRO1387, anti-PRO1419,anti-PRO1433, anti-PRO1474, anti-PRO1550, anti-PRO1571, anti-PRO1572,anti-PRO1759, anti-PRO1904, anti-PRO35193, anti-PRO4341, anti-PRO4348,anti-PRO4369, anti-PRO4381, anti-PRO4407, anti-PRO4425, anti-PRO4985,anti-PRO4989, anti-PRO5737, anti-PRO5800, anti-PRO5993, anti-PRO6017,anti-PRO7174, anti-PRO9744, anti-PRO9821, anti-PRO9852, anti-PRO9873,anti-PRO10196, anti-PRO34778, anti-PRO20233, anti-PRO21956,anti-PRO57290, anti-PRO38465, anti-PRO38683 or anti-PRO85161 antibodies(see below) as long as they exhibit the desired biological orimmunological activity. The term “immunoglobulin” (Ig) is usedinterchangeable with antibody herein.

An “isolated antibody” is one which has been identified and separatedand/or recovered from a component of its natural environment.Contaminant components of its natural environment are materials whichwould interfere with diagnostic or therapeutic uses for the antibody,and may include enzymes, hormones, and other proteinaceous ornonproteinaceous solutes. The invention provides that the antibody willbe purified (1) to greater than 95% by weight of antibody as determinedby the Lowry method, and most preferably more than 99% by weight, (2) toa degree sufficient to obtain at least 15 residues of N-terminal orinternal amino acid sequence by use of a spinning cup sequenator, or (3)to homogeneity by SDS-PAGE under reducing or nonreducing conditionsusing Coomassie blue or, preferably, silver stain. Isolated antibodyincludes the antibody in situ within recombinant cells since at leastone component of the antibody's natural environment will not be present.Ordinarily, however, isolated antibody will be prepared by at least onepurification step.

The basic 4-chain antibody unit is a heterotetrameric glycoproteincomposed of two identical light (L) chains and two identical heavy (H)chains (an IgM antibody consists of 5 of the basic heterotetramer unitalong with an additional polypeptide called J chain, and thereforecontain 10 antigen binding sites, while secreted IgA antibodies canpolymerize to form polyvalent assemblages comprising 2-5 of the basic4-chain units along with J chain). In the case of IgGs, the 4-chain unitis generally about 150,000 daltons. Each L chain is linked to a H chainby one covalent disulfide bond, while the two H chains are linked toeach other by one or more disulfide bonds depending on the H chainisotype. Each H and L chain also has regularly spaced intrachaindisulfide bridges. Each H chain has at the N-terminus, a variable domain(V_(H)) followed by three constant domains (C_(H)) for each of the a andγ chains and four C_(H) domains for μ and ε isotypes. Each L chain hasat the N-terminus, a variable domain (V_(L)) followed by a constantdomain (C_(L)) at its other end. The V_(L) is aligned with the V_(H) andthe C_(L) is aligned with the first constant domain of the heavy chain(C_(H) 1). Particular amino acid residues are believed to form aninterface between the light chain and heavy chain variable domains. Thepairing of a V_(H) and V_(L) together forms a single antigen-bindingsite. For the structure and properties of the different classes ofantibodies, see, e.g., Basic and Clinical Immunology, 8th edition,Daniel P. Stites, Abba I. Terr and Tristram G. Parslow (eds.), Appleton& Lange, Norwalk, Conn., 1994, page 71 and Chapter 6.

The L chain from any vertebrate species can be assigned to one of twoclearly distinct types, called kappa and lambda, based on the amino acidsequences of their constant domains. Depending on the amino acidsequence of the constant domain of their heavy chains (C_(H)),immunoglobulins can be assigned to different classes or isotypes. Thereare five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, havingheavy chains designated α, δ, ε, γ, and μ, respectively. The γ and αclasses are further divided into subclasses on the basis of relativelyminor differences in C_(H) sequence and function, e.g., humans expressthe following subclasses: IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2.

The term “variable” refers to the fact that certain segments of thevariable domains differ extensively in sequence among antibodies. The Vdomain mediates antigen binding and define specificity of a particularantibody for its particular antigen. However, the variability is notevenly distributed across the 110-amino acid span of the variabledomains. Instead, the V regions consist of relatively invariantstretches called framework regions (FRs) of 15-30 amino acids separatedby shorter regions of extreme variability called “hypervariable regions”that are each 9-12 amino acids long. The variable domains of nativeheavy and light chains each comprise four FRs, largely adopting aβ-sheet configuration, connected by three hypervariable regions, whichform loops connecting, and in some cases forming part of, the β-sheetstructure. The hypervariable regions in each chain are held together inclose proximity by the FRs and, with the hypervariable regions from theother chain, contribute to the formation of the antigen-binding site ofantibodies (see Kabat et al., Sequences of Proteins of ImmunologicalInterest, 5th Ed. Public Health Service, National Institutes of Health,Bethesda, Md. (1991)). The constant domains are not involved directly inbinding an antibody to an antigen, but exhibit various effectorfunctions, such as participation of the antibody in antibody dependentcellular cytotoxicity (ADCC).

The term “hypervariable region” when used herein refers to the aminoacid residues of an antibody which are responsible for antigen-binding.The hypervariable region generally comprises amino acid residues from a“complementarity determining region” or “CDR” (e.g. around aboutresidues 24-34 (L1), 50-56 (L2) and 89-97 (L3) in the V_(L), and aroundabout 1-35 (H1), 50-65 (H2) and 95-102 (H3) in the V_(H); Kabat et al.,Sequences of Proteins of Immunological Interest, 5th Ed. Public HealthService, National Institutes of Health, Bethesda, Md. (1991)) and/orthose residues from a “hypervariable loop” (e.g. residues 26-32 (L1),50-52 (L2) and 91-96 (L3) in the V_(L), and 26-32 (H1), 53-55 (H2) and96-101 (H3) in the V_(H); Chothia and Lesk J. Mol. Biol. 196:901-917(1987)).

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. Monoclonal antibodies are highly specific, being directedagainst a single antigenic site. Furthermore, in contrast to polyclonalantibody preparations which include different antibodies directedagainst different determinants (epitopes), each monoclonal antibody isdirected against a single determinant on the antigen. In addition totheir specificity, the monoclonal antibodies are advantageous in thatthey may be synthesized uncontaminated by other antibodies. The modifier“monoclonal” is not to be construed as requiring production of theantibody by any particular method. For example, the monoclonalantibodies useful in the present invention may be prepared by thehybridoma methodology first described by Kohler et al., Nature, 256:495(1975), or may be made using recombinant DNA methods in bacterial,eukaryotic animal or plant cells (see, e.g., U.S. Pat. No. 4,816,567).The “monoclonal antibodies” may also be isolated from phage antibodylibraries using the techniques described in Clackson et al., Nature,352:624-628 (1991) and Marks et al., J. Mol. Biol., 222:581-597 (1991),for example.

The monoclonal antibodies herein include “chimeric” antibodies in whicha portion of the heavy and/or light chain is identical with orhomologous to corresponding sequences in antibodies derived from aparticular species or belonging to a particular antibody class orsubclass, while the remainder of the chain(s) is identical with orhomologous to corresponding sequences in antibodies derived from anotherspecies or belonging to another antibody class or subclass, as well asfragments of such antibodies, so long as they exhibit the desiredbiological activity (see U.S. Pat. No. 4,816,567; and Morrison et al.,Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)). Chimeric antibodies ofinterest herein include “primatized” antibodies comprising variabledomain antigen-binding sequences derived from a non-human primate (e.g.Old World Monkey, Ape etc), and human constant region sequences.

An “intact” antibody is one which comprises an antigen-binding site aswell as a C_(L) and at least heavy chain constant domains, C_(H) 1,C_(H) 2 and C_(H) 3. The constant domains may be native sequenceconstant domains (e.g. human native sequence constant domains) or aminoacid sequence variant thereof. Preferably, the intact antibody has oneor more effector functions.

“Antibody fragments” comprise a portion of an intact antibody,preferably the antigen binding or variable region of the intactantibody. Examples of antibody fragments include Fab, Fab′, F(ab′)₂, andFv fragments; diabodies; linear antibodies (see U.S. Pat. No. 5,641,870,Example 2; Zapata et al., Protein Eng. 8(10): 1057-1062 [1995]);single-chain antibody molecules; and multispecific antibodies formedfrom antibody fragments.

Papain digestion of antibodies produces two identical antigen-bindingfragments, called “Fab” fragments, and a residual “Fc” fragment, adesignation reflecting the ability to crystallize readily. The Fabfragment consists of an entire L chain along with the variable regiondomain of the H chain (V_(H)), and the first constant domain of oneheavy chain (C_(H) 1). Each Fab fragment is monovalent with respect toantigen binding, i.e., it has a single antigen-binding site. Pepsintreatment of an antibody yields a single large F(ab′)₂ fragment whichroughly corresponds to two disulfide linked Fab fragments havingdivalent antigen-binding activity and is still capable of cross-linkingantigen. Fab′ fragments differ from Fab fragments by having additionalfew residues at the carboxy terminus of the C_(H) 1 domain including oneor more cysteines from the antibody hinge region. Fab′-SH is thedesignation herein for Fab′ in which the cysteine residue(s) of theconstant domains bear a free thiol group. F(ab′)₂ antibody fragmentsoriginally were produced as pairs of Fab′ fragments which have hingecysteines between them. Other chemical couplings of antibody fragmentsare also known.

The Fc fragment comprises the carboxy-terminal portions of both H chainsheld together by disulfides. The effector functions of antibodies aredetermined by sequences in the Fc region, which region is also the partrecognized by Fc receptors (FcR) found on certain types of cells.

“Fv” is the minimum antibody fragment which contains a completeantigen-recognition and -binding site. This fragment consists of a dimerof one heavy- and one light-chain variable region domain in tight,non-covalent association. From the folding of these two domains emanatesix hypervariable loops (3 loops each from the H and L chain) thatcontribute the amino acid residues for antigen binding and conferantigen binding specificity to the antibody. However, even a singlevariable domain (or half of an Fv comprising only three CDRs specificfor an antigen) has the ability to recognize and bind antigen, althoughat a lower affinity than the entire binding site.

“Single-chain Fv” also abbreviated as “sFv” or “scFv” are antibodyfragments that comprise the V_(H) and V_(L) antibody domains connectedinto a single polypeptide chain. Preferably, the sFv polypeptide furthercomprises a polypeptide linker between the V_(H) and V_(L) domains whichenables the sFv to form the desired structure for antigen binding. For areview of sFv, see Pluckthun in The Pharmacology of MonoclonalAntibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, NewYork, pp. 269-315 (1994); Borrebaeck 1995, infra.

The term “diabodies” refers to small antibody fragments prepared byconstructing sFv fragments (see preceding paragraph) with short linkers(about 5-10 residues) between the V_(H) and V_(L) domains such thatinter-chain but not intra-chain pairing of the V domains is achieved,resulting in a bivalent fragment, i.e., fragment having twoantigen-binding sites. Bispecific diabodies are heterodimers of two“crossover” sFv fragments in which the V_(H) and V_(L) domains of thetwo antibodies are present on different polypeptide chains. Diabodiesare described more fully in, for example, EP 404,097; WO 93/11161; andHollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993).

“Humanized” forms of non-human (e.g., rodent) antibodies are chimericantibodies that contain minimal sequence derived from the non-humanantibody. For the most part, humanized antibodies are humanimmunoglobulins (recipient antibody) in which residues from ahypervariable region of the recipient are replaced by residues from ahypervariable region of a non-human species (donor antibody) such asmouse, rat, rabbit or non-human primate having the desired antibodyspecificity, affinity, and capability. In some instances, frameworkregion (FR) residues of the human immunoglobulin are replaced bycorresponding non-human residues. Furthermore, humanized antibodies maycomprise residues that are not found in the recipient antibody or in thedonor antibody. These modifications are made to further refine antibodyperformance. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the hypervariable loops correspondto those of a non-human immunoglobulin and all or substantially all ofthe FRs are those of a human immunoglobulin sequence. The humanizedantibody optionally also will comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin. For further details, see Jones et al., Nature321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); andPresta, Curr. Op. Struct. Biol. 2:593-596 (1992).

A “species-dependent antibody,” e.g., a mammalian anti-human IgEantibody, is an antibody which has a stronger binding affinity for anantigen from a first mammalian species than it has for a homologue ofthat antigen from a second mammalian species. Normally, thespecies-dependent antibody “bind specifically” to a human antigen (i.e.,has a binding affinity (Kd) value of no more than about 1×10⁻⁷ M,preferably no more than about 1×10⁻⁸ and most preferably no more thanabout 1×10⁻⁹ M) but has a binding affinity for a homologue of theantigen from a second non-human mammalian species which is at leastabout 50 fold, or at least about 500 fold, or at least about 1000 fold,weaker than its binding affinity for the human antigen. Thespecies-dependent antibody can be of any of the various types ofantibodies as defined above, but preferably is a humanized or humanantibody.

A “PRO226, PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382,PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343,PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571,PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381,PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017,PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196, PRO34778,PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161 bindingoligopeptide” is an oligopeptide that binds, preferably specifically, toa PRO226, PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382,PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343,PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571,PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381,PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017,PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196, PRO34778,PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161 polypeptideas described herein. PRO226, PRO257, PRO268, PRO290, PRO36006, PRO363,PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155,PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474,PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348,PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800,PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196,PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161binding oligopeptides may be chemically synthesized using knownoligopeptide synthesis methodology or may be prepared and purified usingrecombinant technology. PRO226, PRO257, PRO268, PRO290, PRO36006,PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125, PRO1134,PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433,PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341,PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737,PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873,PRO10196, PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 orPRO85161 binding oligopeptides usually are or are at least about 5 aminoacids in length, alternatively are or are at least about 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64,65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82,83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or100 amino acids in length or more, wherein such oligopeptides that arecapable of binding, preferably specifically, to a PRO226, PRO257,PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382, PRO444, PRO705,PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343, PRO1379, PRO1380,PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571, PRO1572, PRO1759,PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381, PRO4407, PRO4425,PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017, PRO7174, PRO9744,PRO9821, PRO9852, PRO9873, PRO10196, PRO34778, PRO20233, PRO21956,PRO57290, PRO38465, PRO38683 or PRO85161 polypeptide as describedherein. PRO226, PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365,PRO382, PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281,PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550,PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369,PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993,PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196,PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161binding oligopeptides may be identified without undue experimentationusing well known techniques. In this regard, it is noted that techniquesfor screening oligopeptide libraries for oligopeptides that are capableof specifically binding to a polypeptide target are well known in theart (see, e.g., U.S. Pat. Nos. 5,556,762, 5,750,373, 4,708,871,4,833,092, 5,223,409, 5,403,484, 5,571,689, 5,663,143; PCT PublicationNos. WO 84/03506 and WO84/03564; Geysen et al., Proc. Natl. Acad. Sci.U.S.A., 81:3998-4002 (1984); Geysen et al., Proc. Natl. Acad. Sci.U.S.A., 82:178-182 (1985); Geysen et al., in Synthetic Peptides asAntigens, 130-149 (1986); Geysen et al., J. Immunol. Meth., 102:259-274(1987); Schoofs et al., J. Immunol., 140:611-616 (1988), Cwirla, S. E.et al. (1990) Proc. Natl. Acad. Sci. USA, 87:6378; Lowman, H. B. et al.(1991) Biochemistry, 30:10832; Clackson, T. et al. (1991) Nature,352:624; Marks, J. D. et al. (1991), J. Mol. Biol., 222:581; Kang, A. S.et al. (1991) Proc. Natl. Acad. Sci. USA, 88:8363, and Smith, G. P.(1991) Current Opin. Biotechnol., 2:668).

A “PRO226, PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382,PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343,PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571,PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381,PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017,PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196, PRO34778,PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161 bindingorganic molecule” is an organic molecule other than an oligopeptide orantibody as defined herein that binds, preferably specifically, to aPRO226, PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382,PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343,PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571,PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381,PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017,PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196, PRO34778,PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161 polypeptideas described herein. PRO226, PRO257, PRO268, PRO290, PRO36006, PRO363,PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155,PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474,PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348,PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800,PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196,PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161binding organic molecules may be identified and chemically synthesizedusing known methodology (see, e.g., PCT Publication Nos. WO00/00823 andWO00/39585). PRO226, PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365,PRO382, PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281,PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550,PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369,PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993,PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196,PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161binding organic molecules are usually less than about 2000 daltons insize, alternatively less than about 1500, 750, 500, 250 or 200 daltonsin size, wherein such organic molecules that are capable of binding,preferably specifically, to a PRO226, PRO257, PRO268, PRO290, PRO36006,PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125, PRO1134,PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433,PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341,PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737,PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873,PRO10196, PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 orPRO85161 polypeptide as described herein may be identified without undueexperimentation using well known techniques. In this regard, it is notedthat techniques for screening organic molecule libraries for moleculesthat are capable of binding to a polypeptide target are well known inthe art (see, e.g., PCT Publication Nos. WO00/00823 and WO00/39585).

An antibody, oligopeptide or other organic molecule “which binds” anantigen of interest, e.g. a tumor-associated polypeptide antigen target,is one that binds the antigen with sufficient affinity such that theantibody, oligopeptide or other organic molecule is preferably useful asa diagnostic and/or therapeutic agent in targeting a cell or tissueexpressing the antigen, and does not significantly cross-react withother proteins. The extent of binding of the antibody, oligopeptide orother organic molecule to a “non-target” protein will be less than about10% of the binding of the antibody, oligopeptide or other organicmolecule to its particular target protein as determined by fluorescenceactivated cell sorting (FACS) analysis or radioimmunoprecipitation(RIA). With regard to the binding of an antibody, oligopeptide or otherorganic molecule to a target molecule, the term “specific binding” or“specifically binds to” or is “specific for” a particular polypeptide oran epitope on a particular polypeptide target means binding that ismeasurably different from a non-specific interaction. Specific bindingcan be measured, for example, by determining binding of a moleculecompared to binding of a control molecule, which generally is a moleculeof similar structure that does not have binding activity. For example,specific binding can be determined by competition with a controlmolecule that is similar to the target, for example, an excess ofnon-labeled target. In this case, specific binding is indicated if thebinding of the labeled target to a probe is competitively inhibited byexcess unlabeled target. The term “specific binding” or “specificallybinds to” or is “specific for” a particular polypeptide or an epitope ona particular polypeptide target as used herein can be exhibited, forexample, by a molecule having a Kd for the target of at least about 10⁻⁴M, alternatively at least about 10⁻⁵ M, alternatively at least about10⁻⁶ M, alternatively at least about 10⁻⁷ M, alternatively at leastabout 10⁻⁸ M, alternatively at least about 10⁻⁹ M, alternatively atleast about 10⁻¹⁰ M, alternatively at least about 10⁻¹¹ M, alternativelyat least about 10⁻¹² M, or greater. The term “specific binding” refersto binding where a molecule binds to a particular polypeptide or epitopeon a particular polypeptide without substantially binding to any otherpolypeptide or polypeptide epitope.

An antibody, oligopeptide or other organic molecule that “inhibits thegrowth of tumor cells expressing a “PRO226, PRO257, PRO268, PRO290,PRO36006, PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125,PRO1134, PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419,PRO1433, PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193,PRO4341, PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989,PRO5737, PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852,PRO9873, PRO10196, PRO34778, PRO20233, PRO21956, PRO57290, PRO38465,PRO38683 or PRO85161?” or a “growth inhibitory” antibody, oligopeptideor other organic molecule is one which results in measurable growthinhibition of cancer cells expressing or overexpressing the appropriatePRO226, PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382,PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343,PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571,PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381,PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017,PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196, PRO34778,PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161polypeptide. The PRO226, PRO257, PRO268, PRO290, PRO36006, PRO363,PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155,PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474,PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348,PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800,PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196,PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161polypeptide may be a transmembrane polypeptide expressed on the surfaceof a cancer cell or may be a polypeptide that is produced and secretedby a cancer cell. Preferred growth inhibitory anti-PRO226, anti-PRO257,anti-PRO268, anti-PRO290, anti-PRO36006, anti-PRO363, anti-PRO365,anti-PRO382, anti-PRO444, anti-PRO705, anti-PRO1071, anti-PRO1125,anti-PRO1134, anti-PRO1155, anti-PRO1281, anti-PRO1343, anti-PRO1379,anti-PRO1380, anti-PRO1387, anti-PRO1419, anti-PRO1433, anti-PRO1474,anti-PRO1550, anti-PRO1571, anti-PRO1572, anti-PRO1759, anti-PRO1904,anti-PRO35193, anti-PRO4341, anti-PRO4348, anti-PRO4369, anti-PRO4381,anti-PRO4407, anti-PRO4425, anti-PRO4985, anti-PRO4989, anti-PRO5737,anti-PRO5800, anti-PRO5993, anti-PRO6017, anti-PRO7174, anti-PRO9744,anti-PRO9821, anti-PRO9852, anti-PRO9873, anti-PRO10196, anti-PRO34778,anti-PRO20233, anti-PRO21956, anti-PRO57290, anti-PRO38465,anti-PRO38683 or anti-PRO85161 antibodies, oligopeptides or organicmolecules inhibit growth of PRO226-, PRO257-, PRO268-, PRO290-,PRO36006-, PRO363-, PRO365-, PRO382-, PRO444-, PRO705-, PRO1071-,PRO1125-, PRO1134-, PRO1155-, PRO1281-, PRO1343-, PRO1379-, PRO1380-,PRO1387-, PRO1419-, PRO1433-, PRO1474-, PRO1550-, PRO1571-, PRO1572-,PRO1759-, PRO1904-, PRO35193-, PRO4341-, PRO4348-, PRO4369-, PRO4381-,PRO4407-, PRO4425-, PRO4985-, PRO4989-, PRO5737-, PRO5800-, PRO5993-,PRO6017-, PRO7174-, PRO9744-, PRO9821-, PRO9852-, PRO9873-, PRO10196-,PRO34778-, PRO20233-, PRO21956-, PRO57290-, PRO38465-, PRO38683- orPRO85161-expressing tumor cells by or by greater than 20%, preferablyfrom about 20% to about 50%, and even more preferably, by or by greaterthan 50% (e.g., from about 50% to about 100%) as compared to theappropriate control, the control typically being tumor cells not treatedwith the antibody, oligopeptide or other organic molecule being tested.Growth inhibition can be measured at an antibody concentration of about0.1 to 30 μg/ml or about 0.5 nM to 200 nM in cell culture, where thegrowth inhibition is determined 1-10 days after exposure of the tumorcells to the antibody. Growth inhibition of tumor cells in vivo can bedetermined in various ways. The antibody is growth inhibitory in vivo ifadministration of the anti-PRO226, anti-PRO257, anti-PRO268,anti-PRO290, anti-PRO36006, anti-PRO363, anti-PRO365, anti-PRO382,anti-PRO444, anti-PRO705, anti-PRO1071, anti-PRO1125, anti-PRO1134,anti-PRO1155, anti-PRO1281, anti-PRO1343, anti-PRO1379, anti-PRO1380,anti-PRO1387, anti-PRO1419, anti-PRO1433, anti-PRO1474, anti-PRO1550,anti-PRO1571, anti-PRO1572, anti-PRO1759, anti-PRO1904, anti-PRO35193,anti-PRO4341, anti-PRO4348, anti-PRO4369, anti-PRO4381, anti-PRO4407,anti-PRO4425, anti-PRO4985, anti-PRO4989, anti-PRO5737, anti-PRO5800,anti-PRO5993, anti-PRO6017, anti-PRO7174, anti-PRO9744, anti-PRO9821,anti-PRO9852, anti-PRO9873, anti-PRO10196, anti-PRO34778, anti-PRO20233,anti-PRO21956, anti-PRO57290, anti-PRO38465, anti-PRO38683 oranti-PRO85161 antibody at about 1 μg/kg to about 100 mg/kg body weightresults in reduction in tumor size or tumor cell proliferation withinabout 5 days to 3 months from the first administration of the antibody,preferably within about 5 to 30 days.

An antibody, oligopeptide or other organic molecule which “inducesapoptosis” is one which induces programmed cell death as determined bybinding of annexin V, fragmentation of DNA, cell shrinkage, dilation ofendoplasmic reticulum, cell fragmentation, and/or formation of membranevesicles (called apoptotic bodies). The cell is usually one whichoverexpresses a PRO226, PRO257, PRO268, PRO290, PRO36006, PRO363,PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155,PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474,PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348,PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800,PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196,PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161polypeptide. Preferably the cell is a tumor cell, e.g., a prostate,breast, ovarian, stomach, endometrial, lung, kidney, colon, bladdercell. Various methods are available for evaluating the cellular eventsassociated with apoptosis. For example, phosphatidyl serine (PS)translocation can be measured by annexin binding; DNA fragmentation canbe evaluated through DNA laddering; and nuclear/chromatin condensationalong with DNA fragmentation can be evaluated by any increase inhypodiploid cells. Preferably, the antibody, oligopeptide or otherorganic molecule which induces apoptosis is one which results in or inabout 2 to 50 fold, preferably in or in about 5 to 50 fold, and mostpreferably in or in about 10 to 50 fold, induction of annexin bindingrelative to untreated cell in an annexin binding assay.

Antibody “effector functions” refer to those biological activitiesattributable to the Fc region (a native sequence Fc region or amino acidsequence variant Fc region) of an antibody, and vary with the antibodyisotype. Examples of antibody effector functions include: C1q bindingand complement dependent cytotoxicity; Fc receptor binding;antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptor); and B cellactivation.

“Antibody-dependent cell-mediated cytotoxicity” or “ADCC” refers to aform of cytotoxicity in which secreted Ig bound onto Fc receptors (FcRs)present on certain cytotoxic cells (e.g., Natural Killer (NK) cells,neutrophils, and macrophages) enable these cytotoxic effector cells tobind specifically to an antigen-bearing target cell and subsequentlykill the target cell with cytotoxins. The antibodies “arm” the cytotoxiccells and are absolutely required for such killing. The primary cellsfor mediating ADCC, NK cells, express FcγRIII only, whereas monocytesexpress FcγRI, FcγRII and FcγRIII. FcR expression on hematopoietic cellsis summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev.Immunol. 9:457-92 (1991). To assess ADCC activity of a molecule ofinterest, an in vitro ADCC assay, such as that described in U.S. Pat.No. 5,500,362 or 5,821,337 may be performed. Useful effector cells forsuch assays include peripheral blood mononuclear cells (PBMC) andNatural Killer (NK) cells. Alternatively, or additionally, ADCC activityof the molecule of interest may be assessed in vivo, e.g., in a animalmodel such as that disclosed in Clynes et al. Proc. Natl. Acad. Sci.U.S.A. 95:652-656 (1998).

“Fc receptor” or “FcR” describes a receptor that binds to the Fc regionof an antibody. The preferred FcR is a native sequence human FcR.Moreover, a preferred FcR is one which binds an IgG antibody (a gammareceptor) and includes receptors of the FcγRI, FcγRII and FcγRIIIsubclasses, including allelic variants and alternatively spliced formsof these receptors. FcγRII receptors include FcγRIIA (an “activatingreceptor”) and FcγRIIB (an “inhibiting receptor”), which have similaramino acid sequences that differ primarily in the cytoplasmic domainsthereof. Activating receptor FcγRIIA contains an immunoreceptortyrosine-based activation motif (ITAM) in its cytoplasmic domain.Inhibiting receptor FcγRIIB contains an immunoreceptor tyrosine-basedinhibition motif (ITIM) in its cytoplasmic domain. (see review M. inDaëron, Annu. Rev. Immunol. 15:203-234 (1997)). FcRs are reviewed inRavetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991); Capel et al.,Immunomethods 4:25-34 (1994); and de Haas et al., J. Lab. Clin. Med.126:330-41 (1995). Other FcRs, including those to be identified in thefuture, are encompassed by the term “FcR” herein. The term also includesthe neonatal receptor, FcRn, which is responsible for the transfer ofmaternal IgGs to the fetus (Guyer et al., J. Immunol. 117:587 (1976) andKim et al., J. Immunol. 24:249 (1994)).

“Human effector cells” are leukocytes which express one or more FcRs andperform effector functions. Preferably, the cells express at leastFcγRIII and perform ADCC effector function. Examples of human leukocyteswhich mediate ADCC include peripheral blood mononuclear cells (PBMC),natural killer (NK) cells, monocytes, cytotoxic T cells and neutrophils;with PBMCs and NK cells being preferred. The effector cells may beisolated from a native source, e.g., from blood.

“Complement dependent cytotoxicity” or “CDC” refers to the lysis of atarget cell in the presence of complement. Activation of the classicalcomplement pathway is initiated by the binding of the first component ofthe complement system (C1q) to antibodies (of the appropriate subclass)which are bound to their cognate antigen. To assess complementactivation, a CDC assay, e.g., as described in Gazzano-Santoro et al.,J. Immunol. Methods 202:163 (1996), may be performed.

The terms “cancer” and “cancerous” refer to or describe thephysiological condition in mammals that is typically characterized byunregulated cell growth. Examples of cancer include but are not limitedto, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. Moreparticular examples of such cancers include squamous cell cancer, lungcancer (including small-cell lung cancer, non-small cell lung cancer,adenocarcinoma of the lung, and squamous carcinoma of the lung), cancerof the peritoneum, hepatocellular cancer, gastric or stomach cancer(including gastrointestinal cancer), pancreatic cancer, glioblastoma,cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma,breast cancer, colon cancer, colorectal cancer, endometrial or uterinecarcinoma, salivary gland carcinoma, kidney or renal cancer, livercancer, prostate cancer, vulval cancer, thyroid cancer, hepaticcarcinoma and various types of head and neck cancer, as well as B-celllymphoma (including low grade/follicular non-Hodgkin's lymphoma (NHL);small lymphocytic (SL) NHL; intermediate grade/follicular NHL;intermediate grade diffuse NHL; high grade immunoblastic NHL; high gradelymphoblastic NHL; high grade small non-cleaved cell NHL; bulky diseaseNHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom'sMacroglobulinemia); chronic lymphocytic leukemia (CLL); acutelymphoblastic leukemia (ALL); Hairy cell leukemia; chronic myeloblasticleukemia; and post-transplant lymphoproliferative disorder (PTLD).Preferably, the cancer comprises a tumor that expresses an IGF receptor,more preferably breast cancer, lung cancer, colorectal cancer, orprostate cancer, and most preferably breast or prostate cancer.

A “chemotherapeutic agent” is a chemical compound useful in thetreatment of cancer. Examples of chemotherapeutic agents includealkylating agents such as thiotepa and CYTOXAN® cyclosphosphamide; alkylsulfonates such as busulfan, improsulfan and piposulfan; aziridines suchas benzodopa, carboquone, meturedopa, and uredopa; ethylenimines andmethylamelamines including altretamine, triethylenemelamine,trietylenephosphoramide, triethiylenethiophosphoramide andtrimethylolomelamine; acetogenins (especially bullatacin andbullatacinone); a camptothecin (including the synthetic analoguetopotecan); bryostatin; callystatin; CC-1065 (including its adozelesin,carzelesin and bizelesin synthetic analogues); cryptophycins(particularly cryptophycin 1 and cryptophycin 8); dolastatin;duocarmycin (including the synthetic analogues, KW-2189 and CB1-TM1);eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogenmustards such as chlorambucil, chlomaphazine, cholophosphamide,estramustine, ifosfamide, mechlorethamine, mechlorethamine oxidehydrochloride, melphalan, novembichin, phenesterine, prednimustine,trofosfamide, uracil mustard; nitrosureas such as carmustine,chlorozotocin, fotemustine, lomustine, nimustine, and ranimustine;antibiotics such as the enediyne antibiotics (e.g., calicheamicin,especially calicheamicin gamma1I and calicheamicin omegaI1 (see, e.g.,Agnew, Chem. Intl. Ed. Engl., 33: 183-186 (1994)); dynemicin, includingdynemicin A; bisphosphonates, such as clodronate; an esperamicin; aswell as neocarzinostatin chromophore and related chromoprotein enediyneantiobiotic chromophores), aclacinomysins, actinomycin, authramycin,azaserine, bleomycins, cactinomycin, carabicin, caminomycin,carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin,6-diazo-5-oxo-L-norleucine, ADRIAMYCIN® doxorubicin (includingmorpholino-doxorubicin, cyanomorpholino-doxorubicin,2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin,idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolicacid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin,quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin,ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexateand 5-fluorouracil (5-FU); folic acid analogues such as denopterin,methotrexate, pteropterin, trimetrexate; purine analogs such asfludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogs such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine;androgens such as calusterone, dromostanolone propionate, epitiostanol,mepitiostane, testolactone; anti-adrenals such as aminoglutethimide,mitotane, trilostane; folic acid replenisher such as frolinic acid;aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil;amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine;diaziquone; elfomithine; elliptinium acetate; an epothilone; etoglucid;gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids suchas maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol;nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone;podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK® polysaccharidecomplex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin;sizofuran; spirogermanium; tenuazonic acid; triaziquone;2,2′,2″-trichlorotriethylamine; trichothecenes (especially T-2 toxin,verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine;mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxoids, e.g., TAXOLpaclitaxel (Bristol-Myers Squibb Oncology, Princeton, N.J.), ABRAXANE™Cremophor-free, albumin-engineered nanoparticle formulation ofpaclitaxel (American Pharmaceutical Partners, Schaumberg, Ill.), andTAXOTERE doxetaxel (Rhône-Poulenc Rorer, Antony, France); chloranbucil;GEMZAR® gemcitabine; 6-thioguanine; mercaptopurine; methotrexate;platinum analogs such as cisplatin and carboplatin; vinblastine;platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine;NAVELBINE vinorelbine; novantrone; teniposide; edatrexate; daunomycin;aminopterin; xeloda; ibandronate; CPT-11; topoisomerase inhibitor RFS2000; difluoromethylomithine (DMFO); retinoids such as retinoic acid;capecitabine; and pharmaceutically acceptable salts, acids orderivatives of any of the above.

Also included in this definition are anti-hormonal agents that act toregulate or inhibit hormone action on tumors such as anti-estrogens andselective estrogen receptor modulators (SERMs), including, for example,tamoxifen (including NOLVADEX® tamoxifen), raloxifene, droloxifene,4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, andFARESTON• toremifene; aromatase inhibitors that inhibit the enzymearomatase, which regulates estrogen production in the adrenal glands,such as, for example, 4(5)-imidazoles, aminoglutethimide, MEGASE®megestrol acetate, AROMASIN® exemestane, formestanie, fadrozole,RIVISOR® vorozole, FEMARA® letrozole, and ARIMIDEX® anastrozole; andanti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide,and goserelin; as well as troxacitabine (a 1,3-dioxolane nucleosidecytosine analog); antisense oligonucleotides, particularly those whichinhibit expression of genes in signaling pathways implicated in abherantcell proliferation, such as, for example, PKC-alpha, Ralf and H-Ras;ribozymes such as a VEGF expression inhibitor (e.g., ANGIOZYME®ribozyme) and a HER2 expression inhibitor; vaccines such as gene therapyvaccines, for example, ALLOVECTIN® vaccine, LEUVECTIN® vaccine, andVAXID® vaccine; PROLEUKIN® rIL-2; LURTOTECAN® topoisomerase 1 inhibitor;ABARELIX® rmRH; and pharmaceutically acceptable salts, acids orderivatives of any of the above.

The terms “cell proliferative disorder” and “proliferative disorder”refer to disorders that are associated with some degree of abnormal cellproliferation. In one aspect of the invention, the cell proliferativedisorder is cancer.

“Tumor”, as used herein, refers to all neoplastic cell growth andproliferation, whether malignant or benign, and all pre-cancerous andcancerous cells and tissues.

An antibody, oligopeptide or other organic molecule which “induces celldeath” is one which causes a viable cell to become nonviable. The cellis one which expresses a PRO226, PRO257, PRO268, PRO290, PRO36006,PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125, PRO1134,PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433,PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341,PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737,PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873,PRO10196, PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 orPRO85161 polypeptide, preferably a cell that overexpresses a PRO226,PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382, PRO444,PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343, PRO1379,PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571, PRO1572,PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381, PRO4407,PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017, PRO7174,PRO9744, PRO9821, PRO9852, PRO9873, PRO10196, PRO34778, PRO20233,PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161 polypeptide ascompared to a normal cell of the same tissue type. The PRO226, PRO257,PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382, PRO444, PRO705,PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343, PRO1379, PRO1380,PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571, PRO1572, PRO1759,PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381, PRO4407, PRO4425,PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017, PRO7174, PRO9744,PRO9821, PRO9852, PRO9873, PRO10196, PRO34778, PRO20233, PRO21956,PRO57290, PRO38465, PRO38683 or PRO85161 polypeptide may be atransmembrane polypeptide expressed on the surface of a cancer cell ormay be a polypeptide that is produced and secreted by a cancer cell.Preferably, the cell is a cancer cell, e.g., a breast, ovarian, stomach,endometrial, salivary gland, lung, kidney, colon, thyroid, pancreatic orbladder cell. Cell death in vitro may be determined in the absence ofcomplement and immune effector cells to distinguish cell death inducedby antibody-dependent cell-mediated cytotoxicity (ADCC) or complementdependent cytotoxicity (CDC). Thus, the assay for cell death may beperformed using heat inactivated serum (i.e., in the absence ofcomplement) and in the absence of immune effector cells. To determinewhether the antibody, oligopeptide or other organic molecule is able toinduce cell death, loss of membrane integrity as evaluated by uptake ofpropidium iodide (PI), trypan blue (see Moore et al. Cytotechnology17:1-11 (1995)) or 7AAD can be assessed relative to untreated cells.Preferred cell death-inducing antibodies, oligopeptides or other organicmolecules are those which induce PI uptake in the PI uptake assay inBT474 cells.

As used herein, the term “immunoadhesion” designates antibody-likemolecules which combine the binding specificity of a heterologousprotein (an “adhesion”) with the effector functions of immunoglobulinconstant domains. Structurally, the immunoadhesions comprise a fusion ofan amino acid sequence with the desired binding specificity which isother than the antigen recognition and binding site of an antibody(i.e., is “heterologous”), and an immunoglobulin constant domainsequence. The adhesion part of an immunoadhesion molecule typically is acontiguous amino acid sequence comprising at least the binding site of areceptor or a ligand. The immunoglobulin constant domain sequence in theimmunoadhesion may be obtained from any immunoglobulin, such as IgG-1,IgG-2, IgG-3, or IgG-4 subtypes, IgA (including IgA-1 and IgA-2), IgE,IgD or IgM.

The word “label” when used herein refers to a detectable compound orcomposition which is conjugated directly or indirectly to the antibodyso as to generate a “labeled” antibody. The label may be detectable byitself (e.g. radioisotope labels or fluorescent labels) or, in the caseof an enzymatic label, may catalyze chemical alteration of a substratecompound or composition which is detectable.

“Replication-preventing agent” is an agent wherein replication,function, and/or growth of the cells is inhibited or prevented, or cellsare destroyed, no matter what the mechanism, such as by apoptosis,angiostasis, cytosis, tumoricide, mytosis inhibition, blocking cellcycle progression, arresting cell growth, binding to tumors, acting ascellular mediators, etc. Such agents include a chemotherapeutic agent,cytotoxic agent, cytokine, growth-inhibitory agent, or anti-hormonalagent, e.g., an anti-estrogen compound such as tamoxifen, ananti-progesterone such as onapristone (see, EP 616 812); or ananti-androgen such as flutamide, as well as aromidase inhibitors, or ahormonal agent such as an androgen.

The term “cytotoxic agent” as used herein refers to a substance thatinhibits or prevents the function of cells and/or causes destruction ofcells. The term is intended to include radioactive isotopes (e.g.,At²¹¹, I¹³¹, I¹²⁵, Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³, Bi²¹², P³² and radioactiveisotopes of Lu), chemotherapeutic agents e.g. methotrexate, adriamicin,vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin,melphalan, mitomycin C, chlorambucil, daunorubicin or otherintercalating agents, enzymes and fragments thereof such as nucleolyticenzymes, antibiotics, and toxins such as small molecule toxins orenzymatically active toxins of bacterial, fungal, plant or animalorigin, including fragments and/or variants thereof, and the variousantitumor or anticancer agents disclosed below. Other cytotoxic agentsare described below. A tumoricidal agent causes destruction of tumorcells.

Preferred cytotoxic agents herein for the specific tumor types to use incombination with the antagonists herein are as follows:

1. Prostate cancer: androgens, docetaxel, paclitaxel, estramustine,doxorubicin, mitoxantrone, antibodies to ErbB2 domain(s) such as 2C4(WO01/00245; hybridoma ATCC HB-12697), which binds to a region in theextracellular domain of ErbB2 (e.g., any one or more residues in theregion from about residue 22 to about residue 584 of ErbB2, inclusive),AVASTIN™ anti-vascular endothelial growth factor (VEGF), TARCEVA™OSI-774 (erlotinib) (Genenetech and OSI Pharmaceuticals), or otherepidermal growth factor receptor tyrosine kinase inhibitors (EGFRTKI's).2. Stomach cancer: 5-fluorouracil (SFU), XELODA™ capecitabine,methotrexate, etoposide, cisplatin/carboplatin, pacliitaxel, docetaxel,gemcitabine, doxorubicin, and CPT-11 (camptothcin-11; irinotecan, USABrand Name: CAMPTOSAR®).3. Pancreatic cancer: gemcitabine, SFU, XELODA™ capecitabine, CPT-11,docetaxel, paclitaxel, cisplatin, carboplatin, TARCEVA™ erlotinib, andother EGFR TKI's.4. Colorectal cancer: SFU, XELODA™ capecitabine, CPT-11, oxaliplatin,AVASTIN™ anti-VEGF, TARCEVA™ erlotinib and other EGFR TKI's, andERBITUX™ (formerly known as IMC-C225) human:murine-chimerized monoclonalantibody that binds to EGFR and blocks the ability of EGF to initiatereceptor activation and signaling to the tumor.5. Renal cancer: IL-2, interferon alpha, AVASTIN™ anti-VEGF, MEGACE™(Megestrol acetate) progestin, vinblastine, TARCEVA™ erlotinib, andother EGFR TKI's.

A “growth inhibitory agent” when used herein refers to a compound orcomposition which inhibits growth of a cell, especially a PRO226-,PRO257-, PRO268-, PRO290-, PRO36006-, PRO363-, PRO365-, PRO382-,PRO444-, PRO705-, PRO1071-, PRO1125-, PRO1134-, PRO1155-, PRO1281-,PRO1343-, PRO1379-, PRO1380-, PRO1387-, PRO1419-, PRO1433-, PRO1474-,PRO1550-, PRO1571-, PRO1572-, PRO1759-, PRO1904-, PRO35193-, PRO4341-,PRO4348-, PRO4369-, PRO4381-, PRO4407-, PRO4425-, PRO4985-, PRO4989-,PRO5737-, PRO5800-, PRO5993-, PRO6017-, PRO7174-, PRO9744-, PRO9821-,PRO9852-, PRO9873-, PRO10196-, PRO34778-, PRO20233-, PRO21956-,PRO57290-, PRO38465-, PRO38683- or PRO85161-expressing cancer cell,either in vitro or in vivo. Thus, the growth inhibitory agent may be onewhich significantly reduces the percentage of PRO226-, PRO257-, PRO268-,PRO290-, PRO36006-, PRO363-, PRO365-, PRO382-, PRO444-, PRO705-,PRO1071-, PRO1125-, PRO1134-, PRO155-, PRO1281-, PRO1343-, PRO1379-,PRO1380-, PRO1387-, PRO1419-, PRO1433-, PRO1474-, PRO1550-, PRO1571-,PRO1572-, PRO1759-, PRO1904-, PRO35193-, PRO4341-, PRO4348-, PRO4369-,PRO4381-, PRO4407-, PRO4425-, PRO4985-, PRO4989-, PRO5737-, PRO5800-,PRO5993-, PRO6017-, PRO7174-, PRO9744-, PRO9821-, PRO9852-, PRO9873-,PRO10196-, PRO34778-, PRO20233-, PRO21956-, PRO57290-, PRO38465-,PRO38683- or PRO85161-expressing cells in S phase. Examples of growthinhibitory agents include agents that block cell cycle progression (at aplace other than S phase), such as agents that induce G1 arrest andM-phase arrest. Classical M-phase blockers include the vincas(vincristine and vinblastine), taxanes, and topoisomerase II inhibitorssuch as doxorubicin, epirubicin, daunorubicin, etoposide, and bleomycin.Those agents that arrest G1 also spill over into S-phase arrest, forexample, DNA alkylating agents such as tamoxifen, prednisone,dacarbazine, mechlorethamine, cisplatin, methotrexate, 5-fluorouracil,and ara-C. Further information can be found in The Molecular Basis ofCancer, Mendelsohn and Israel, eds., Chapter 1, entitled “Cell cycleregulation, oncogenes, and antineoplastic drugs” by Murakami et al. (WBSaunders: Philadelphia, 1995), especially p. 13. The taxanes (paclitaxeland docetaxel) are anticancer drugs both derived from the yew tree.Docetaxel (TAXOTERE, Rhone-Poulenc Rorer), derived from the Europeanyew, is a semisynthetic analogue of paclitaxel (TAXOL®, Bristol-MyersSquibb). Paclitaxel and docetaxel promote the assembly of microtubulesfrom tubulin dimers and stabilize microtubules by preventingdepolymerization, which results in the inhibition of mitosis in cells.

“Doxorubicin” is an anthracycline antibiotic. The full chemical name ofdoxorubicin is(8S-cis)-10-[(3-amino-2,3,6-trideoxy-α-L-lyxo-hexapyranosyl)oxy]-7,8,9,10-tetrahydro-6,8,11-trihydroxy-8-(hydroxyacetyl)-1-methoxy-5,12-naphthacenedione.

The term “cytokine” is a generic term for proteins released by one cellpopulation which act on another cell as intercellular mediators.Examples of such cytokines are lymphokines, monokines, and traditionalpolypeptide hormones. Included among the cytokines are growth hormonesuch as human growth hormone, N-methionyl human growth hormone, andbovine growth hormone; parathyroid hormone; thyroxine; insulin;proinsulin; relaxin; prorelaxin; glycoprotein hormones such as folliclestimulating hormone (FSH), thyroid stimulating hormone (TSH), andluteinizing hormone (LH); hepatic growth factor; fibroblast growthfactor; prolactin; placental lactogen; tumor necrosis factor-α and -β;mullerian-inhibiting substance; mouse gonadotropin-associated peptide;inhibin; activin; vascular endothelial growth factor; integrin;thrombopoietin (TPO); nerve growth factors such as NGF-β;platelet-growth factor; transforming growth factors (TGFs) such as TGF-αand TGF-β; insulin-like growth factor-I and -II; erythropoietin (EPO);osteoinductive factors; interferons such as interferon-α, -β, and -γ;colony stimulating factors (CSFs) such as macrophage-CSF (M-CSF);granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF);interleukins (ILs) such as IL-1, IL-1a, IL-2, IL-3, IL-4, IL-5, IL-6,IL-7, IL-8, IL-9, IL-11, IL-12; a tumor necrosis factor such as TNF-α orTNF-β; and other polypeptide factors including LIF and kit ligand (KL).As used herein, the term cytokine includes proteins from natural sourcesor from recombinant cell culture and biologically active equivalents ofthe native sequence cytokines.

The term “package insert” is used to refer to instructions customarilyincluded in commercial packages of therapeutic products, that containinformation about the indications, usage, dosage, administration,contraindications and/or warnings concerning the use of such therapeuticproducts.

The term “gene” refers to (a) a gene containing at least one of the DNAsequences disclosed herein; (b) any DNA sequence that encodes the aminoacid sequence encoded by the DNA sequences disclosed herein and/or; (c)any DNA sequence that hybridizes to the complement of the codingsequences disclosed herein. Preferably, the term includes coding as wellas noncoding regions, and preferably includes all sequences necessaryfor normal gene expression.

The term “gene targeting” refers to a type of homologous recombinationthat occurs when a fragment of genomic DNA is introduced into amammalian cell and that fragment locates and recombines with endogenoushomologous sequences. Gene targeting by homologous recombination employsrecombinant DNA technologies to replace specific genomic sequences withexogenous DNA of particular design.

The term “homologous recombination” refers to the exchange of DNAfragments between two DNA molecules or chromatids at the site ofhomologous nucleotide sequences.

The term “target gene” (alternatively referred to as “target genesequence” or “target DNA sequence”) refers to any nucleic acid molecule,polynucleotide, or gene to be modified by homologous recombination. Thetarget sequence includes an intact gene, an exon or intron, a regulatorysequence or any region between genes. The target gene my comprise aportion of a particular gene or genetic locus in the individual'sgenomic DNA.

“Disruption” of a PRO226, PRO257, PRO268, PRO290, PRO36006, PRO363,PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155,PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474,PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348,PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800,PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196,PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161gene occurs when a fragment of genomic DNA locates and recombines withan endogenous homologous sequence wherein the disruption is a deletionof the native gene or a portion thereof, or a mutation in the nativegene or wherein the disruption is the functional inactivation of thenative gene. Alternatively, sequence disruptions may be generated bynonspecific insertional inactivation using a gene trap vector (i.e.non-human transgenic animals containing and expressing a randomlyinserted transgene; see for example U.S. Pat. No. 6,436,707 issued Aug.20, 2002). These sequence disruptions or modifications may includeinsertions, missense, frameshift, deletion, or substitutions, orreplacements of DNA sequence, or any combination thereof. Insertionsinclude the insertion of entire genes, which may be of animal, plant,fungal, insect, prokaryotic, or viral origin. Disruption, for example,can alter the normal gene product by inhibiting its production partiallyor completely or by enhancing the normal gene product's activity.Preferably, the disruption is a null disruption, wherein there is nosignificant expression of the PRO226, PRO257, PRO268, PRO290, PRO36006,PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125, PRO1134,PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433,PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341,PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737,PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873,PRO10196, PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 orPRO85161 gene.

The term “native expression” refers to the expression of the full-lengthpolypeptide encoded by the PRO226, PRO257, PRO268, PRO290, PRO36006,PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125, PRO1134,PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433,PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341,PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737,PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873,PRO10196, PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 orPRO85161 gene, at expression levels present in the wild-type mouse.Thus, a disruption in which there is “no native expression” of theendogenous PRO226, PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365,PRO382, PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281,PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550,PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369,PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993,PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196,PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161gene refers to a partial or complete reduction of the expression of atleast a portion of a polypeptide encoded by an endogenous PRO226,PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382, PRO444,PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343, PRO1379,PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571, PRO1572,PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381, PRO4407,PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017, PRO7174,PRO9744, PRO9821, PRO9852, PRO9873, PRO10196, PRO34778, PRO20233,PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161 gene of a singlecell, selected cells, or all of the cells of a mammal.

The term “knockout” refers to the disruption of a PRO226, PRO257,PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382, PRO444, PRO705,PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343, PRO1379, PRO1380,PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571, PRO1572, PRO1759,PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381, PRO4407, PRO4425,PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017, PRO7174, PRO9744,PRO9821, PRO9852, PRO9873, PRO10196, PRO34778, PRO20233, PRO21956,PRO57290, PRO38465, PRO38683 or PRO85161 gene wherein the disruptionresults in: the functional inactivation of the native gene; the deletionof the native gene or a portion thereof; or a mutation in the nativegene.

The term “knock-in” refers to the replacement of the mouse ortholog (orother mouse gene) with a human cDNA encoding any of the specific humanPRO226-, PRO257-, PRO268-, PRO290-, PRO36006-, PRO363-, PRO365-,PRO382-, PRO444-, PRO705-, PRO1071-, PRO1125-, PRO1134-, PRO1155-,PRO1281-, PRO1343-, PRO1379-, PRO1380-, PRO1387-, PRO1419-, PRO1433-,PRO1474-, PRO1550-, PRO1571-, PRO1572-, PRO1759-, PRO1904-, PRO35193-,PRO4341-, PRO4348-, PRO4369-, PRO4381-, PRO4407-, PRO4425-, PRO4985-,PRO4989-, PRO5737-, PRO5800-, PRO5993-, PRO6017-, PRO7174-, PRO9744-,PRO9821-, PRO9852-, PRO9873-, PRO10196-, PRO34778-, PRO20233-,PRO21956-, PRO57290-, PRO38465-, PRO38683- or PRO85161-encoding genes orvariants thereof (ie. the disruption results in are placement of anative mouse gene with a native human gene).

The term “construct” or “targeting construct” refers to an artificiallyassembled DNA segment to be transferred into a target tissue, cell lineor animal. Typically, the targeting construct will include a gene or anucleic acid sequence of particular interest, a marker gene andappropriate control sequences. As provided herein, the targetingconstruct comprises a PRO226, PRO257, PRO268, PRO290, PRO36006, PRO363,PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155,PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474,PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348,PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800,PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196,PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161targeting construct. A “PRO226, PRO257, PRO268, PRO290, PRO36006,PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125, PRO1134,PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433,PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341,PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737,PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873,PRO10196, PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 orPRO85161 targeting construct” includes a DNA sequence homologous to atleast one portion of a PRO226, PRO257, PRO268, PRO290, PRO36006, PRO363,PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155,PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474,PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348,PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800,PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196,PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161gene and is capable of producing a disruption in a PRO226, PRO257,PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382, PRO444, PRO705,PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343, PRO1379, PRO1380,PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571, PRO1572, PRO1759,PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381, PRO4407, PRO4425,PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017, PRO7174, PRO9744,PRO9821, PRO9852, PRO9873, PRO10196, PRO34778, PRO20233, PRO21956,PRO57290, PRO38465, PRO38683 or PRO85161 gene in a host cell.

The term “transgenic cell” refers to a cell containing within its genomea PRO226, PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382,PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343,PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571,PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381,PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017,PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196, PRO34778,PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161 gene thathas been disrupted, modified, altered, or replaced completely orpartially by the method of gene targeting.

The term “transgenic animal” refers to an animal that contains withinits genome a specific gene that has been disrupted or otherwise modifiedor mutated by the methods described herein or methods otherwise wellknown in the art. Preferably the non-human transgenic animal is amammal. More preferably, the mammal is a rodent such as a rat or mouse.In addition, a “transgenic animal” may be a heterozygous animal (i.e.,one defective allele and one wild-type allele) or a homozygous animal(i.e., two defective alleles). An embryo is considered to fall withinthe definition of an animal. The provision of an animal includes theprovision of an embryo or foetus in utero, whether by mating orotherwise, and whether or not the embryo goes to term.

As used herein, the terms “selective marker” and position selectionmarker” refer to a gene encoding a product that enables only the cellsthat carry the gene to survive and/or grow under certain conditions. Forexample, plant and animal cells that express the introduced neomycinresistance (Neo^(r)) gene are resistant to the compound G418. Cells thatdo not carry the Neo^(r) gene marker are killed by G418. Other positiveselection markers are known to, or are within the purview of, those ofordinary skill in the art.

The term “modulates” or “modulation” as used herein refers to thedecrease, inhibition, reduction, amelioration, increase or enhancementof a PRO226, PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382,PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343,PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571,PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381,PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017,PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196, PRO34778,PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161 genefunction, expression, activity, or alternatively a phenotype associatedwith PRO226, PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382,PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343,PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571,PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381,PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017,PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196, PRO34778,PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161 gene.

The term “ameliorates” or “amelioration” as used herein refers to adecrease, reduction or elimination of a condition, disease, disorder, orphenotype, including an abnormality or symptom.

The term “abnormality” refers to any disease, disorder, condition, orphenotype in which PRO226, PRO257, PRO268, PRO290, PRO36006, PRO363,PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155,PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474,PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348,PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800,PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196,PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161is implicated, including pathological conditions and behavioralobservations.

TABLE 2 PRO XXXXXXXXXXXXXXX (Length = 15 amino acids) ComparisonXXXXXYYYYYYY (Length = 12 amino acids) Protein % amino acid sequenceidentity = (the number of identically matching amino acid residuesbetween the two polypeptide sequences as determined by ALIGN-2) dividedby (the total number of amino acid residues of the PRO polypeptide) = 5divided by 15 = 33.3%

TABLE 3 PRO XXXXXXXXXX (Length = 10 amino acids) ComparisonXXXXXYYYYYYZZYZ (Length = 15 amino acids) Protein % amino acid sequenceidentity = (the number of identically matching amino acid residuesbetween the two polypeptide sequences as determined by ALIGN-2) dividedby (the total number of amino acid residues of the PRO polypeptide) = 5divided by 10 = 50%

TABLE 4 PRO-DNA NNNNNNNNNNNNNN (Length = 14 nucleotides) ComparisonNNNNNNLLLLLLLLLL (Length = 16 nucleotides) DNA % nucleic acid sequenceidentity = (the number of identically matching nucleotides between thetwo nucleic acid sequences as determined by ALIGN-2) divided by (thetotal number of nucleotides of the PRO-DNA nucleic acid sequence) = 6divided by 14 = 42.9%

TABLE 5 PRO-DNA NNNNNNNNNNNN (Length = 12 nucleotides) Comparison DNANNNNLLLVV (Length = 9 nucleotides) % nucleic acid sequence identity =(the number of identically matching nucleotides between the two nucleicacid sequences as determined by ALIGN-2) divided by (the total number ofnucleotides of the PRO-DNA nucleic acid sequence) = 4 divided by 12 =33.3%

II. Compositions and Methods of the Invention

A. Full-Length PRO226, PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365,PRO382, PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281,PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550,PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369,PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993,PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196,PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161Polypeptides

The present invention provides newly identified and isolated nucleotidesequences encoding polypeptides referred to in the present applicationas PRO226, PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382,PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343,PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571,PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381,PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017,PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196, PRO34778,PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161polypeptides. In particular, cDNAs encoding various PRO226, PRO257,PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382, PRO444, PRO705,PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343, PRO1379, PRO1380,PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571, PRO1572, PRO1759,PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381, PRO4407, PRO4425,PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017, PRO7174, PRO9744,PRO9821, PRO9852, PRO9873, PRO10196, PRO34778, PRO20233, PRO21956,PRO57290, PRO38465, PRO38683 or PRO85161 polypeptides have beenidentified and isolated, as disclosed in further detail in the Examplesbelow. It is noted that proteins produced in separate expression roundsmay be given different PRO numbers but the UNQ number is unique for anygiven DNA and the encoded protein, and will not be changed. However, forsake of simplicity, in the present specification the protein encoded bythe full length native nucleic acid molecules disclosed herein as wellas all further native homologues and variants included in the foregoingdefinition of PRO, will be referred to as “PRO/number”, regardless oftheir origin or mode of preparation.

As disclosed in the Examples below, various cDNA clones have beendeposited with the ATCC. The actual nucleotide sequences of those clonescan readily be determined by the skilled artisan by sequencing of thedeposited clone using routine methods in the art. The predicted aminoacid sequence can be determined from the nucleotide sequence usingroutine skill. For the PRO226, PRO257, PRO268, PRO290, PRO36006, PRO363,PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155,PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474,PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348,PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800,PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196,PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161polypeptides and encoding nucleic acids described herein, Applicantshave identified what is believed to be the reading frame bestidentifiable with the sequence information available at the time.

B. PRO226, PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382,PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343,PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571,PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381,PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017,PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196, PRO34778,PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161 PolypeptideVariants

In addition to the full-length native sequence PRO226, PRO257, PRO268,PRO290, PRO36006, PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071,PRO1125, PRO1134, PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387,PRO1419, PRO1433, PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904,PRO35193, PRO4341, PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985,PRO4989, PRO5737, PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821,PRO9852, PRO9873, PRO10196, PRO34778, PRO20233, PRO21956, PRO57290,PRO38465, PRO38683 or PRO85161 polypeptides described herein, it iscontemplated that PRO226, PRO257, PRO268, PRO290, PRO36006, PRO363,PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155,PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474,PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348,PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800,PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196,PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161variants can be prepared. PRO226, PRO257, PRO268, PRO290, PRO36006,PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125, PRO1134,PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433,PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341,PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737,PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873,PRO10196, PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 orPRO85161 variants can be prepared by introducing appropriate nucleotidechanges into the PRO226, PRO257, PRO268, PRO290, PRO36006, PRO363,PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155,PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474,PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348,PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800,PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196,PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161DNA, and/or by synthesis of the desired PRO226, PRO257, PRO268, PRO290,PRO36006, PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125,PRO1134, PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419,PRO1433, PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193,PRO4341, PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989,PRO5737, PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852,PRO9873, PRO10196, PRO34778, PRO20233, PRO21956, PRO57290, PRO38465,PRO38683 or PRO85161 polypeptide. Those skilled in the art willappreciate that amino acid changes may alter post-translationalprocesses of the PRO226, PRO257, PRO268, PRO290, PRO36006, PRO363,PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155,PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474,PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348,PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800,PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196,PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161polypeptide, such as changing the number or position of glycosylationsites or altering the membrane anchoring characteristics.

Variations in the native full-length sequence PRO226, PRO257, PRO268,PRO290, PRO36006, PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071,PRO1125, PRO1134, PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387,PRO1419, PRO1433, PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904,PRO35193, PRO4341, PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985,PRO4989, PRO5737, PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821,PRO9852, PRO9873, PRO10196, PRO34778, PRO20233, PRO21956, PRO57290,PRO38465, PRO38683 or PRO85161 polypeptide or in various domains of thePRO226, PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382,PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343,PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571,PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381,PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017,PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196, PRO34778,PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161 polypeptidedescribed herein, can be made, for example, using any of the techniquesand guidelines for conservative and non-conservative mutations setforth, for instance, in U.S. Pat. No. 5,364,934. Variations may be asubstitution, deletion or insertion of one or more codons encoding thePRO226, PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382,PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343,PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571,PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381,PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017,PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196, PRO34778,PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161 polypeptidethat results in a change in the amino acid sequence of the PRO226,PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382, PRO444,PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343, PRO1379,PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571, PRO1572,PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381, PRO4407,PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017, PRO7174,PRO9744, PRO9821, PRO9852, PRO9873, PRO10196, PRO34778, PRO20233,PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161 polypeptide ascompared with the native sequence PRO226, PRO257, PRO268, PRO290,PRO36006, PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125,PRO1134, PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419,PRO1433, PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193,PRO4341, PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989,PRO5737, PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852,PRO9873, PRO10196, PRO34778, PRO20233, PRO21956, PRO57290, PRO38465,PRO38683 or PRO85161 polypeptide. Optionally the variation is bysubstitution of at least one amino acid with any other amino acid in oneor more of the domains of the PRO226, PRO257, PRO268, PRO290, PRO36006,PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125, PRO1134,PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433,PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341,PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737,PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873,PRO10196, PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 orPRO85161 polypeptide. Guidance in determining which amino acid residuemay be inserted, substituted or deleted without adversely affecting thedesired activity may be found by comparing the sequence of the PRO226,PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382, PRO444,PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343, PRO1379,PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571, PRO1572,PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381, PRO4407,PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017, PRO7174,PRO9744, PRO9821, PRO9852, PRO9873, PRO10196, PRO34778, PRO20233,PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161 polypeptide with thatof homologous known protein molecules and minimizing the number of aminoacid sequence changes made in regions of high homology. Amino acidsubstitutions can be the result of replacing one amino acid with anotheramino acid having similar structural and/or chemical properties, such asthe replacement of a leucine with a serine, i.e., conservative aminoacid replacements. Insertions or deletions may optionally be in therange of about 1 to 5 amino acids. The variation allowed may bedetermined by systematically making insertions, deletions orsubstitutions of amino acids in the sequence and testing the resultingvariants for activity exhibited by the full-length or mature nativesequence.

PRO226, PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382,PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343,PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571,PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381,PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017,PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196, PRO34778,PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161 polypeptidefragments are provided herein. Such fragments may be truncated at theN-terminus or C-terminus, or may lack internal residues, for example,when compared with a full length native protein. Certain fragments lackamino acid residues that are not essential for a desired biologicalactivity of the PRO226, PRO257, PRO268, PRO290, PRO36006, PRO363,PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155,PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474,PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348,PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800,PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196,PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161polypeptide.

PRO226, PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382,PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343,PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571,PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381,PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017,PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196, PRO34778,PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161 fragmentsmay be prepared by any of a number of conventional techniques. Desiredpeptide fragments may be chemically synthesized. An alternative approachinvolves generating PRO226, PRO257, PRO268, PRO290, PRO36006, PRO363,PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155,PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474,PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348,PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800,PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196,PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161fragments by enzymatic digestion, e.g., by treating the protein with anenzyme known to cleave proteins at sites defined by particular aminoacid residues, or by digesting the DNA with suitable restriction enzymesand isolating the desired fragment. Yet another suitable techniqueinvolves isolating and amplifying a DNA fragment encoding a desiredpolypeptide fragment, by polymerase chain reaction (PCR).Oligonucleotides that define the desired termini of the DNA fragment areemployed at the 5′ and 3′ primers in the PCR. Preferably, PRO226,PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382, PRO444,PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343, PRO1379,PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571, PRO1572,PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381, PRO4407,PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017, PRO7174,PRO9744, PRO9821, PRO9852, PRO9873, PRO10196, PRO34778, PRO20233,PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161 polypeptide fragmentsshare at least one biological and/or immunological activity with thenative PRO226, PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382,PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343,PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571,PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381,PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017,PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196, PRO34778,PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161 polypeptidedisclosed herein.

Conservative substitutions of interest are shown in Table 6 under theheading of preferred substitutions. If such substitutions result in achange in biological activity, then more substantial changes,denominated exemplary substitutions in Table 6, or as further describedbelow in reference to amino acid classes, are preferably introduced andthe products screened.

TABLE 6 Original Exemplary Preferred Residue Substitutions SubstitutionsAla (A) Val; Leu; Ile Val Arg ®) Lys; Gln; Asn Lys Asn (N) Gln; His;Asp, Lys; Arg Gln Asp (D) Glu; Asn Glu Cys ©) Ser; Ala Ser Gln (Q) Asn;Glu Asn Glu (E) Asp; Gln Asp Gly (G) Ala Ala His (H) Asn; Gln; Lys; ArgArg Ile (I) Leu; Val; Met; Ala; Leu Phe; Norleucine Leu (L) Norleucine;Ile; Val; Ile Met; Ala; Phe Lys (K) Arg; Gln; Asn Arg Met (M) Leu; Phe;Ile Leu Phe (F) Trp; Leu; Val; Ile; Ala; Tyr Tyr Pro (P) Ala Ala Ser (S)Thr Thr Thr (T) Val; Ser Ser Trp (W) Tyr; Phe Tyr Tyr (Y) Trp; Phe; Thr;Ser Phe Val (V) Ile; Leu; Met; Phe; Leu Ala; Norleucine

Substantial modifications in function or immunological identity of thePRO226, PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382,PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343,PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571,PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381,PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017,PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196, PRO34778,PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161 polypeptideare accomplished by selecting substitutions that differ significantly intheir effect on maintaining (a) the structure of the polypeptidebackbone in the area of the substitution, for example, as a sheet orhelical conformation, (b) the charge or hydrophobicity of the moleculeat the target site, or (c) the bulk of the side chain. Naturallyoccurring residues are divided into groups based on common side-chainproperties: Amino acids may be grouped according to similarities in theproperties of their side chains (in A. L. Lehninger, in Biochemistry,second ed., pp. 73-75, Worth Publishers, New York (1975)):

(1) non-polar: Ala (A), Val (V), Leu (L), Ile (I), Pro (P), Phe (F), Trp(W), Met (M)

(2) uncharged polar: Gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y), Asn(N), Gln (O)

(3) acidic: Asp (D), Glu (E)

(4) basic: Lys (K), Arg (R), H is (H)

Alternatively, naturally occurring residues may be divided into groupsbased on common side-chain properties:

(1) hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile;

(2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;

(3) acidic: Asp, Glu;

(4) basic: His, Lys, Arg;

(5) residues that influence chain orientation: Gly, Pro;

(6) aromatic: Trp, Tyr, Phe.

Non-conservative substitutions will entail exchanging a member of one ofthese classes for another class. Such substituted residues also may beintroduced into the conservative substitution sites or, more preferably,into the remaining (non-conserved) sites.

The variations can be made using methods known in the art such asoligonucleotide-mediated (site-directed) mutagenesis, alanine scanning,and PCR mutagenesis. Site-directed mutagenesis [Carter et al., Nucl.Acids Res., 13:4331 (1986); Zoller et al., Nucl. Acids Res., 10:6487(1987)], cassette mutagenesis [Wells et al., Gene, 34:315 (1985)],restriction selection mutagenesis [Wells et al., Philos. Trans. R. Soc.London SerA, 317:415 (1986)] or other known techniques can be performedon the cloned DNA to produce the PRO226, PRO257, PRO268, PRO290,PRO36006, PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125,PRO1134, PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419,PRO1433, PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193,PRO4341, PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989,PRO5737, PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852,PRO9873, PRO10196, PRO34778, PRO20233, PRO21956, PRO57290, PRO38465,PRO38683 or PRO85161 variant DNA.

Scanning amino acid analysis can also be employed to identify one ormore amino acids along a contiguous sequence. Among the preferredscanning amino acids are relatively small, neutral amino acids. Suchamino acids include alanine, glycine, serine, and cysteine. Alanine istypically a preferred scanning amino acid among this group because iteliminates the side-chain beyond the beta-carbon and is less likely toalter the main-chain conformation of the variant [Cunningham and Wells,Science, 244: 1081-1085 (1989)]. Alanine is also typically preferredbecause it is the most common amino acid. Further, it is frequentlyfound in both buried and exposed positions [Creighton, The Proteins,(W.H. Freeman & Co., N.Y.); Chothia, J. Mol. Biol., 150:1 (1976)]. Ifalanine substitution does not yield adequate amounts of variant, anisoteric amino acid can be used.

C. Modifications of PRO226, PRO257, PRO268, PRO290, PRO36006, PRO363,PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155,PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474,PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348,PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800,PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196,PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161Polypeptides

Covalent modifications of PRO226, PRO257, PRO268, PRO290, PRO36006,PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125, PRO1134,PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433,PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341,PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737,PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873,PRO10196, PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 orPRO85161 polypeptides are included within the scope of this invention.One type of covalent modification includes reacting targeted amino acidresidues of a PRO226, PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365,PRO382, PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281,PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550,PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369,PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993,PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196,PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161polypeptide with an organic derivatizing agent that is capable ofreacting with selected side chains or the N or C-terminal residues ofthe PRO226, PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382,PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343,PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571,PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381,PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017,PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196, PRO34778,PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161polypeptide. Derivatization with bifunctional agents is useful, forinstance, for crosslinking PRO226, PRO257, PRO268, PRO290, PRO36006,PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125, PRO1134,PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433,PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341,PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737,PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873,PRO10196, PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 orPRO85161 polypeptides to a water-insoluble support matrix or surface foruse in the method for purifying anti-PRO226, anti-PRO257, anti-PRO268,anti-PRO290, anti-PRO36006, anti-PRO363, anti-PRO365, anti-PRO382,anti-PRO444, anti-PRO705, anti-PRO1071, anti-PRO1125, anti-PRO1134,anti-PRO1155, anti-PRO1281, anti-PRO1343, anti-PRO1379, anti-PRO1380,anti-PRO1387, anti-PRO1419, anti-PRO1433, anti-PRO1474, anti-PRO1550,anti-PRO1571, anti-PRO1572, anti-PRO1759, anti-PRO1904, anti-PRO35193,anti-PRO4341, anti-PRO4348, anti-PRO4369, anti-PRO4381, anti-PRO4407,anti-PRO4425, anti-PRO4985, anti-PRO4989, anti-PRO5737, anti-PRO5800,anti-PRO5993, anti-PRO6017, anti-PRO7174, anti-PRO9744, anti-PRO9821,anti-PRO9852, anti-PRO9873, anti-PRO10196, anti-PRO34778, anti-PRO20233,anti-PRO21956, anti-PRO57290, anti-PRO38465, anti-PRO38683 oranti-PRO85161 antibodies, and vice-versa. Commonly used crosslinkingagents include, e.g., 1,1-bis(diazoacetyl)-2-phenylethane,glutaraldehyde, N-hydroxysuccinimide esters, for example, esters with4-azidosalicylic acid, homobifunctional imidoesters, includingdisuccinimidyl esters such as 3,3′-dithiobis(succinimidylpropionate),bifunctional maleimides such as bis-N-maleimido-1,8-octane and agentssuch as methyl-3-[(p-azidophenyl)dithio]propioimidate.

Other modifications include deamidation of glutaminyl and asparaginylresidues to the corresponding glutamyl and aspartyl residues,respectively, hydroxylation of proline and lysine, phosphorylation ofhydroxyl groups of seryl or threonyl residues, methylation of theα-amino groups of lysine, arginine, and histidine side chains [T. E.Creighton, Proteins: Structure and Molecular Properties, W.H. Freeman &Co., San Francisco, pp. 79-86 (1983)], acetylation of the N-terminalamine, and amidation of any C-terminal carboxyl group.

Another type of covalent modification of the PRO226, PRO257, PRO268,PRO290, PRO36006, PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071,PRO1125, PRO1134, PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387,PRO1419, PRO1433, PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904,PRO35193, PRO4341, PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985,PRO4989, PRO5737, PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821,PRO9852, PRO9873, PRO10196, PRO34778, PRO20233, PRO21956, PRO57290,PRO38465, PRO38683 or PRO85161 polypeptide included within the scope ofthis invention comprises altering the native glycosylation pattern ofthe polypeptide. “Altering the native glycosylation pattern” is intendedfor purposes herein to mean deleting one or more carbohydrate moietiesfound in native sequence PRO226, PRO257, PRO268, PRO290, PRO36006,PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125, PRO1134,PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433,PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341,PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737,PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873,PRO10196, PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 orPRO85161 polypeptides (either by removing the underlying glycosylationsite or by deleting the glycosylation by chemical and/or enzymaticmeans), and/or adding one or more glycosylation sites that are notpresent in the native sequence PRO226, PRO257, PRO268, PRO290, PRO36006,PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125, PRO1134,PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433,PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341,PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737,PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873,PRO10196, PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 orPRO85161 polypeptide. In addition, the phrase includes qualitativechanges in the glycosylation of the native proteins, involving a changein the nature and proportions of the various carbohydrate moietiespresent.

Addition of glycosylation sites to the PRO226, PRO257, PRO268, PRO290,PRO36006, PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125,PRO1134, PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419,PRO1433, PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193,PRO4341, PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989,PRO5737, PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852,PRO9873, PRO10196, PRO34778, PRO20233, PRO21956, PRO57290, PRO38465,PRO38683 or PRO85161 polypeptide may be accomplished by altering theamino acid sequence. The alteration may be made, for example, by theaddition of, or substitution by, one or more serine or threonineresidues to the native sequence PRO226, PRO257, PRO268, PRO290,PRO36006, PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125,PRO1134, PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419,PRO1433, PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193,PRO4341, PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989,PRO5737, PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852,PRO9873, PRO10196, PRO34778, PRO20233, PRO21956, PRO57290, PRO38465,PRO38683 or PRO85161 (for O-linked glycosylation sites). The PRO226,PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382, PRO444,PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343, PRO1379,PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571, PRO1572,PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381, PRO4407,PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017, PRO7174,PRO9744, PRO9821, PRO9852, PRO9873, PRO10196, PRO34778, PRO20233,PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161 amino acid sequencemay optionally be altered through changes at the DNA level, particularlyby mutating the DNA encoding the PRO226, PRO257, PRO268, PRO290,PRO36006, PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125,PRO1134, PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419,PRO1433, PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193,PRO4341, PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989,PRO5737, PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852,PRO9873, PRO10196, PRO34778, PRO20233, PRO21956, PRO57290, PRO38465,PRO38683 or PRO85161 polypeptide at preselected bases such that codonsare generated that will translate into the desired amino acids.

Another means of increasing the number of carbohydrate moieties on thePRO226, PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382,PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343,PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571,PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381,PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017,PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196, PRO34778,PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161 polypeptideis by chemical or enzymatic coupling of glycosides to the polypeptide.Such methods are described in the art, e.g., in WO 87/05330 published 11Sep. 1987, and in Aplin and Wriston, CRC Crit. Rev. Biochem., pp.259-306 (1981).

Removal of carbohydrate moieties present on the PRO226, PRO257, PRO268,PRO290, PRO36006, PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071,PRO1125, PRO1134, PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387,PRO1419, PRO1433, PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904,PRO35193, PRO4341, PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985,PRO4989, PRO5737, PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821,PRO9852, PRO9873, PRO10196, PRO34778, PRO20233, PRO21956, PRO57290,PRO38465, PRO38683 or PRO85161 polypeptide may be accomplishedchemically or enzymatically or by mutational substitution of codonsencoding for amino acid residues that serve as targets forglycosylation. Chemical deglycosylation techniques are known in the artand described, for instance, by Hakimuddin, et al., Arch. Biochem.Biophys., 259:52 (1987) and by Edge et al., Anal. Biochem., 118:131(1981). Enzymatic cleavage of carbohydrate moieties on polypeptides canbe achieved by the use of a variety of endo- and exo-glycosidases asdescribed by Thotakura et al., Meth. Enzymol., 138:350 (1987).

Another type of covalent modification of PRO226, PRO257, PRO268, PRO290,PRO36006, PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125,PRO1134, PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419,PRO1433, PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193,PRO4341, PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989,PRO5737, PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852,PRO9873, PRO10196, PRO34778, PRO20233, PRO21956, PRO57290, PRO38465,PRO38683 or PRO85161 polypeptides comprises linking the PRO226, PRO257,PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382, PRO444, PRO705,PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343, PRO1379, PRO1380,PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571, PRO1572, PRO1759,PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381, PRO4407, PRO4425,PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017, PRO7174, PRO9744,PRO9821, PRO9852, PRO9873, PRO10196, PRO34778, PRO20233, PRO21956,PRO57290, PRO38465, PRO38683 or PRO85161 polypeptide to one of a varietyof nonproteinaceous polymers, e.g., polyethylene glycol (PEG),polypropylene glycol, or polyoxyalkylenes, in the manner set forth inU.S. Pat. No. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or4,179,337.

The PRO226, PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382,PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343,PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571,PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381,PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017,PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196, PRO34778,PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161polypeptides of the present invention may also be modified in a way toform a chimeric molecule comprising the PRO226, PRO257, PRO268, PRO290,PRO36006, PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125,PRO1134, PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419,PRO1433, PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193,PRO4341, PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989,PRO5737, PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852,PRO9873, PRO10196, PRO34778, PRO20233, PRO21956, PRO57290, PRO38465,PRO38683 or PRO85161 polypeptide fused to another, heterologouspolypeptide or amino acid sequence.

Such a chimeric molecule comprises a fusion of the PRO226, PRO257,PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382, PRO444, PRO705,PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343, PRO1379, PRO1380,PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571, PRO1572, PRO1759,PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381, PRO4407, PRO4425,PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017, PRO7174, PRO9744,PRO9821, PRO9852, PRO9873, PRO10196, PRO34778, PRO20233, PRO21956,PRO57290, PRO38465, PRO38683 or PRO85161 polypeptide with a tagpolypeptide which provides an epitope to which an anti-tag antibody canselectively bind. The epitope tag is generally placed at the amino- orcarboxyl-terminus of the PRO226, PRO257, PRO268, PRO290, PRO36006,PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125, PRO1134,PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433,PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341,PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737,PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873,PRO10196, PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 orPRO85161 polypeptide. The presence of such epitope-tagged forms of thePRO226, PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382,PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343,PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571,PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381,PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017,PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196, PRO34778,PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161 polypeptidecan be detected using an antibody against the tag polypeptide. Also,provision of the epitope tag enables the PRO226, PRO257, PRO268, PRO290,PRO36006, PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125,PRO1134, PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419,PRO1433, PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193,PRO4341, PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989,PRO5737, PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852,PRO9873, PRO10196, PRO34778, PRO20233, PRO21956, PRO57290, PRO38465,PRO38683 or PRO85161 polypeptide to be readily purified by affinitypurification using an anti-tag antibody or another type of affinitymatrix that binds to the epitope tag. Various tag polypeptides and theirrespective antibodies are well known in the art. Examples includepoly-histidine (poly-his) or poly-histidine-glycine (poly-his-gly) tags;the flu HA tag polypeptide and its antibody 12CA5 [Field et al., Mol.Cell. Biol., 8:2159-2165 (1988)]; the c-myc tag and the 8F9, 3C7, 6E10,G4, B7 and 9E10 antibodies thereto [Evan et al., Molecular and CellularBiology, 5:3610-3616 (1985)]; and the Herpes Simplex virus glycoproteinD (gD) tag and its antibody [Paborsky et al., Protein Engineering,3(6):547-553 (1990)]. Other tag polypeptides include the Flag-peptide[Hopp et al., BioTechnology, 6:1204-1210 (1988)]; the KT3 epitopepeptide [Martin et al., Science, 255:192-194 (1992)]; an α-tubulinepitope peptide [Skinner et al., J. Biol. Chem., 266:15163-15166(1991)]; and the T7 gene 10 protein peptide tag [Lutz-Freyermuth et al.,Proc. Natl. Acad. Sci. USA, 87:6393-6397 (1990)].

The chimeric molecule may comprise a fusion of the PRO226, PRO257,PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382, PRO444, PRO705,PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343, PRO1379, PRO1380,PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571, PRO1572, PRO1759,PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381, PRO4407, PRO4425,PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017, PRO7174, PRO9744,PRO9821, PRO9852, PRO9873, PRO10196, PRO34778, PRO20233, PRO21956,PRO57290, PRO38465, PRO38683 or PRO85161 polypeptide with animmunoglobulin or a particular region of an immunoglobulin. For abivalent form of the chimeric molecule (also referred to as an“immunoadhesin”), such a fusion could be to the Fc region of an IgGmolecule. The Ig fusions preferably include the substitution of asoluble (transmembrane domain deleted or inactivated) form of a PRO226,PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382, PRO444,PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343, PRO1379,PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571, PRO1572,PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381, PRO4407,PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017, PRO7174,PRO9744, PRO9821, PRO9852, PRO9873, PRO10196, PRO34778, PRO20233,PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161 polypeptide in placeof at least one variable region within an Ig molecule. In a particularlypreferred aspect of the invention, the immunoglobulin fusion includesthe hinge, CH2 and CH3, or the hinge, CH1, CH2 and CH3 regions of anIgG1 molecule. For the production of immunoglobulin fusions see alsoU.S. Pat. No. 5,428,130 issued Jun. 27, 1995.

D. Preparation of PRO226, PRO257, PRO268, PRO290, PRO36006, PRO363,PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155,PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474,PRO550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348,PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800,PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196,PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161Polypeptides

The description below relates primarily to production of PRO226, PRO257,PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382, PRO444, PRO705,PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343, PRO1379, PRO1380,PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571, PRO1572, PRO1759,PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381, PRO4407, PRO4425,PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017, PRO7174, PRO9744,PRO9821, PRO9852, PRO9873, PRO10196, PRO34778, PRO20233, PRO21956,PRO57290, PRO38465, PRO38683 or PRO85161 polypeptides by culturing cellstransformed or transfected with a vector containing PRO226, PRO257,PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382, PRO444, PRO705,PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343, PRO1379, PRO1380,PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571, PRO1572, PRO1759,PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381, PRO4407, PRO4425,PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017, PRO7174, PRO9744,PRO9821, PRO9852, PRO9873, PRO10196, PRO34778, PRO20233, PRO21956,PRO57290, PRO38465, PRO38683 or PRO85161 nucleic acid. It is, of course,contemplated that alternative methods, which are well known in the art,may be employed to prepare PRO226, PRO257, PRO268, PRO290, PRO36006,PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125, PRO1134,PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433,PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341,PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737,PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873,PRO10196, PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 orPRO85161 polypeptides. For instance, the PRO226, PRO257, PRO268, PRO290,PRO36006, PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125,PRO1134, PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419,PRO1433, PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193,PRO4341, PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989,PRO5737, PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852,PRO9873, PRO10196, PRO34778, PRO20233, PRO21956, PRO57290, PRO38465,PRO38683 or PRO85161 sequence, or portions thereof, may be produced bydirect peptide synthesis using solid-phase techniques [see, e.g.,Stewart et al., Solid-Phase Peptide Synthesis, W.H. Freeman Co., SanFrancisco, Calif. (1969); Merrifield, J. Am. Chem. Soc., 85:2149-2154(1963)]. In vitro protein synthesis may be performed using manualtechniques or by automation. Automated synthesis may be accomplished,for instance, using an Applied Biosystems Peptide Synthesizer (FosterCity, Calif.) using manufacturer's instructions. Various portions of thePRO226, PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382,PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343,PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571,PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381,PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017,PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196, PRO34778,PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161 polypeptidemay be chemically synthesized separately and combined using chemical orenzymatic methods to produce the full-length PRO226, PRO257, PRO268,PRO290, PRO36006, PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071,PRO1125, PRO1134, PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387,PRO1419, PRO1433, PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904,PRO35193, PRO4341, PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985,PRO4989, PRO5737, PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821,PRO9852, PRO9873, PRO10196, PRO34778, PRO20233, PRO21956, PRO57290,PRO38465, PRO38683 or PRO85161 polypeptide.

1. Isolation of DNA Encoding PRO226, PRO257, PRO268, PRO290, PRO36006,PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125, PRO1134,PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433,PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341,PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737,PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873,PRO10196, PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 orPRO85161 Polypeptides

DNA encoding PRO226, PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365,PRO382, PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281,PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550,PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369,PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993,PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196,PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161polypeptides may be obtained from a cDNA library prepared from tissuebelieved to possess the PRO226, PRO257, PRO268, PRO290, PRO36006,PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125, PRO1134,PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433,PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341,PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737,PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873,PRO10196, PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 orPRO85161 mRNA and to express it at a detectable level. Accordingly,human PRO226-, PRO257-, PRO268-, PRO290-, PRO36006-, PRO363-, PRO365-,PRO382-, PRO444-, PRO705-, PRO1071-, PRO1125-, PRO1134-, PRO1155-,PRO1281-, PRO1343-, PRO1379-, PRO1380-, PRO1387-, PRO1419-, PRO1433-,PRO1474-, PRO1550-, PRO1571-, PRO1572-, PRO1759-, PRO1904-, PRO35193-,PRO4341-, PRO4348-, PRO4369-, PRO4381-, PRO4407-, PRO4425-, PRO4985-,PRO4989-, PRO5737-, PRO5800-, PRO5993-, PRO6017-, PRO7174-, PRO9744-,PRO9821-, PRO9852-, PRO9873-, PRO10196-, PRO34778-, PRO20233-,PRO21956-, PRO57290-, PRO38465-, PRO38683- or PRO85161-DNA can beconveniently obtained from a cDNA library prepared from human tissue,such as described in the Examples. The PRO226-, PRO257-, PRO268-,PRO290-, PRO36006-, PRO363-, PRO365-, PRO382-, PRO444-, PRO705-,PRO1071-, PRO1125-, PRO1134-, PRO1155-, PRO1281-, PRO1343-, PRO1379-,PRO1380-, PRO1387-, PRO1419-, PRO1433-, PRO1474-, PRO1550-, PRO1571-,PRO1572-, PRO1759-, PRO1904-, PRO35193-, PRO4341-, PRO4348-, PRO4369-,PRO4381-, PRO4407-, PRO4425-, PRO4985-, PRO4989-, PRO5737-, PRO5800-,PRO5993-, PRO6017-, PRO7174-, PRO9744-, PRO9821-, PRO9852-, PRO9873-,PRO10196-, PRO34778-, PRO20233-, PRO21956-, PRO57290-, PRO38465-,PRO38683 or PRO85161-encoding gene may also be obtained from a genomiclibrary or by known synthetic procedures (e.g., automated nucleic acidsynthesis).

Libraries can be screened with probes (such as antibodies to the PRO226,PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382, PRO444,PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343, PRO1379,PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571, PRO1572,PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381, PRO4407,PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017, PRO7174,PRO9744, PRO9821, PRO9852, PRO9873, PRO10196, PRO34778, PRO20233,PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161 polypeptide oroligonucleotides of at least about 20-80 bases) designed to identify thegene of interest or the protein encoded by it. Screening the cDNA orgenomic library with the selected probe may be conducted using standardprocedures, such as described in Sambrook et al., Molecular Cloning: ALaboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989).An alternative means to isolate the gene encoding PRO226, PRO257,PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382, PRO444, PRO705,PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343, PRO1379, PRO1380,PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571, PRO1572, PRO1759,PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381, PRO4407, PRO4425,PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017, PRO7174, PRO9744,PRO9821, PRO9852, PRO9873, PRO10196, PRO34778, PRO20233, PRO21956,PRO57290, PRO38465, PRO38683 or PRO85161 is to use PCR methodology[Sambrook et al., supra; Dieffenbach et al., PCR Primer: A LaboratoryManual (Cold Spring Harbor Laboratory Press, 1995)].

The Examples below describe techniques for screening a cDNA library. Theoligonucleotide sequences selected as probes should be of sufficientlength and sufficiently unambiguous that false positives are minimized.The oligonucleotide is preferably labeled such that it can be detectedupon hybridization to DNA in the library being screened. Methods oflabeling are well known in the art, and include the use of radiolabelslike ³²P-labeled ATP, biotinylation or enzyme labeling. Hybridizationconditions, including moderate stringency and high stringency, areprovided in Sambrook et al., supra.

Sequences identified in such library screening methods can be comparedand aligned to other known sequences deposited and available in publicdatabases such as GenBank or other private sequence databases. Sequenceidentity (at either the amino acid or nucleotide level) within definedregions of the molecule or across the full-length sequence can bedetermined using methods known in the art and as described herein.

Nucleic acid having protein coding sequence may be obtained by screeningselected cDNA or genomic libraries using the deduced amino acid sequencedisclosed herein for the first time, and, if necessary, usingconventional primer extension procedures as described in Sambrook etal., supra, to detect precursors and processing intermediates of mRNAthat may not have been reverse-transcribed into cDNA.

2. Selection and Transformation of Host Cells

Host cells are transfected or transformed with expression or cloningvectors described herein for PRO226, PRO257, PRO268, PRO290, PRO36006,PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125, PRO1134,PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433,PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341,PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737,PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873,PRO10196, PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 orPRO85161 polypeptide production and cultured in conventional nutrientmedia modified as appropriate for inducing promoters, selectingtransformants, or amplifying the genes encoding the desired sequences.The culture conditions, such as media, temperature, pH and the like, canbe selected by the skilled artisan without undue experimentation. Ingeneral, principles, protocols, and practical techniques for maximizingthe productivity of cell cultures can be found in Mammalian CellBiotechnology: a Practical Approach, M. Butler, ed. (IRL Press, 1991)and Sambrook et al., supra.

Methods of eukaryotic cell transfection and prokaryotic celltransformation are known to the ordinarily skilled artisan, for example,CaCl₂, CaPO₄, liposome-mediated and electroporation. Depending on thehost cell used, transformation is performed using standard techniquesappropriate to such cells. The calcium treatment employing calciumchloride, as described in Sambrook et al., supra, or electroporation isgenerally used for prokaryotes. Infection with Agrobacterium tumefaciensis used for transformation of certain plant cells, as described by Shawet al., Gene, 23:315 (1983) and WO 89/05859 published 29 Jun. 1989. Formammalian cells without such cell walls, the calcium phosphateprecipitation method of Graham and van der Eb, Virology, 52:456-457(1978) can be employed. General aspects of mammalian cell host systemtransfections have been described in U.S. Pat. No. 4,399,216.Transformations into yeast are typically carried out according to themethod of Van Solingen et al., J. Bact., 130:946 (1977) and Hsiao etal., Proc. Natl. Acad. Sci. (USA), 76:3829 (1979). However, othermethods for introducing DNA into cells, such as by nuclearmicroinjection, electroporation, bacterial protoplast fusion with intactcells, or polycations, e.g., polybrene, polyornithine, may also be used.For various techniques for transforming mammalian cells, see Keown etal., Methods in Enzymology, 185:527-537 (1990) and Mansour et al.,Nature, 336:348-352 (1988).

Suitable host cells for cloning or expressing the DNA in the vectorsherein include prokaryote, yeast, or higher eukaryote cells. Suitableprokaryotes include but are not limited to eubacteria, such asGram-negative or Gram-positive organisms, for example,Enterobacteriaceae such as E. coli. Various E. coli strains are publiclyavailable, such as E. coli K12 strain MM294 (ATCC 31,446); E. coli X1776(ATCC 31,537); E. coli strain W3110 (ATCC 27,325) and K5 772 (ATCC53,635). Other suitable prokaryotic host cells includeEnterobacteriaceae such as Escherichia, e.g., E. coli, Enterobacter,Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium,Serratia, e.g., Serratia marcescans, and Shigella, as well as Bacillisuch as B. subtilis and B. licheniformis (e.g., B. licheniformis 41Pdisclosed in DD 266,710 published 12 Apr. 1989), Pseudomonas such as P.aeruginosa, and Streptomyces. These examples are illustrative ratherthan limiting. Strain W3110 is one particularly preferred host or parenthost because it is a common host strain for recombinant DNA productfermentations. Preferably, the host cell secretes minimal amounts ofproteolytic enzymes. For example, strain W3110 may be modified to effecta genetic mutation in the genes encoding proteins endogenous to thehost, with examples of such hosts including E. coli W3110 strain 1A2,which has the complete genotype tonA; E. coli W3110 strain 9E4, whichhas the complete genotype to tonA ptr3; E. coli W3110 strain 27C7 (ATCC55,244), which has the complete genotype tonA ptr3phoA E15 (argF-lac)169degP ompT kan^(r) ; E. coli W3110 strain 37D6, which has the completegenotype tonA ptr3phoA E15 (argF-lac)169 degP ompT rbs7 ilvG kan^(r) ;E. coli W3110 strain 40B4, which is strain 37D6 with a non-kanamycinresistant degP deletion mutation; and an E. coli strain having mutantperiplasmic protease disclosed in U.S. Pat. No. 4,946,783 issued 7 Aug.1990. Alternatively, in vitro methods of cloning, e.g., PCR or othernucleic acid polymerase reactions, are suitable.

In addition to prokaryotes, eukaryotic microbes such as filamentousfingi or yeast are suitable cloning or expression hosts for PRO226-,PRO257-, PRO268-, PRO290-, PRO36006-, PRO363-, PRO365-, PRO382-,PRO444-, PRO705-, PRO1071-, PRO1125-, PRO1134-, PRO1155-, PRO1281-,PRO1343-, PRO1379-, PRO1380-, PRO1387-, PRO1419-, PRO1433-, PRO1474-,PRO1550-, PRO1571-, PRO1572-, PRO1759-, PRO1904-, PRO35193-, PRO4341-,PRO4348-, PRO4369-, PRO4381-, PRO4407-, PRO4425-, PRO4985-, PRO4989-,PRO5737-, PRO5800-, PRO5993-, PRO6017-, PRO7174-, PRO9744-, PRO9821-,PRO9852-, PRO9873-, PRO10196-, PRO34778-, PRO20233-, PRO21956-,PRO57290-, PRO38465-, PRO38683- or PRO85161-encoding vectors.Saccharomyces cerevisiae is a commonly used lower eukaryotic hostmicroorganism. Others include Schizosaccharomyces pombe (Beach andNurse, Nature, 290: 140 [1981]; EP 139,383 published 2 May 1985);Kluyveromyces hosts (U.S. Pat. No. 4,943,529; Fleer et al.,Bio/Technology, 9:968-975 (1991)) such as, e.g., K. lactis (MW98-8C,CBS683, CBS4574; Louvencourt et al., J. Bacteriol., 154(2):737-742[1983]), K. fragilis (ATCC 12,424), K. bulgaricus (ATCC 16,045), K.wickeramii (ATCC 24,178), K. waltii (ATCC 56,500), K. drosophilarum(ATCC 36,906; VandenBerg et al., Bio/Technology, 8:135 (1990)), K.thermotolerans, and K. marxianus; yarrowia (EP 402,226); Pichia pastoris(EP 183,070; Sreekrishna et al., J. Basic Microbiol., 28:265-278[1988]); Candida; Trichoderma reesia (EP 244,234); Neurospora crassa(Case et al., Proc. Natl. Acad. Sci. USA, 76:5259-5263 [1979]);Schwanniomyces such as Schwanniomyces occidentalis (EP 394,538 published31 Oct. 1990); and filamentous fungi such as, e.g., Neurospora,Penicillium, Tolypocladium (WO 91/00357 published 10 Jan. 1991), andAspergillus hosts such as A. nidulans (Ballance et al., Biochem.Biophys. Res. Commun., 112:284-289 [1983]; Tilburn et al., Gene,26:205-221 [1983]; Yelton et al., Proc. Natl. Acad. Sci. USA, 81:1470-1474 [1984]) and A. niger (Kelly and Hynes, EMBO J., 4:475-479[1985]). Methylotropic yeasts are suitable herein and include, but arenot limited to, yeast capable of growth on methanol selected from thegenera consisting of Hansenula, Candida, Kloeckera, Pichia,Saccharomyces, Torulopsis, and Rhodotorula. A list of specific speciesthat are exemplary of this class of yeasts may be found in C. Anthony,The Biochemistry of Methylotrophs, 269 (1982).

Suitable host cells for the expression of glycosylated PRO226, PRO257,PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382, PRO444, PRO705,PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343, PRO1379, PRO1380,PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571, PRO1572, PRO1759,PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381, PRO4407, PRO4425,PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017, PRO7174, PRO9744,PRO9821, PRO9852, PRO9873, PRO10196, PRO34778, PRO20233, PRO21956,PRO57290, PRO38465, PRO38683 or PRO85161 polypeptides are derived frommulticellular organisms. Examples of invertebrate cells include insectcells such as Drosophila S2 and Spodoptera Sf9, as well as plant cells.Examples of useful mammalian host cell lines include Chinese hamsterovary (CHO) and COS cells. More specific examples include monkey kidneyCV1 line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonickidney line (293 or 293 cells subcloned for growth in suspensionculture, Graham et al., J. Gen Virol., 36:59 (1977)); Chinese hamsterovary cells/−DHFR (CHO, Urlaub and Chasin, Proc. Natl. Acad. Sci. USA,77:4216 (1980)); mouse sertoli cells (TM4, Mather, Biol. Reprod.,23:243-251 (1980)); human lung cells (W138, ATCC CCL 75); human livercells (Hep G2, HB 8065); and mouse mammary tumor (MMT 060562, ATCCCCL51). The selection of the appropriate host cell is deemed to bewithin the skill in the art.

3. Selection and Use of a Replicable Vector

The nucleic acid (e.g., cDNA or genomic DNA) encoding PRO226, PRO257,PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382, PRO444, PRO705,PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343, PRO1379, PRO1380,PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571, PRO1572, PRO1759,PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381, PRO4407, PRO4425,PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017, PRO7174, PRO9744,PRO9821, PRO9852, PRO9873, PRO10196, PRO34778, PRO20233, PRO21956,PRO57290, PRO38465, PRO38683 or PRO85161 polypeptides may be insertedinto a replicable vector for cloning (amplification of the DNA) or forexpression. Various vectors are publicly available. The vector may, forexample, be in the form of a plasmid, cosmid, viral particle, or phage.The appropriate nucleic acid sequence may be inserted into the vector bya variety of procedures. In general, DNA is inserted into an appropriaterestriction endonuclease site(s) using techniques known in the art.Vector components generally include, but are not limited to, one or moreof a signal sequence, an origin of replication, one or more markergenes, an enhancer element, a promoter, and a transcription terminationsequence. Construction of suitable vectors containing one or more ofthese components employs standard ligation techniques which are known tothe skilled artisan.

The PRO226, PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382,PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343,PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571,PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381,PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017,PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196, PRO34778,PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161 polypeptidemay be produced recombinantly not only directly, but also as a fusionpolypeptide with a heterologous polypeptide, which may be a signalsequence or other polypeptide having a specific cleavage site at theN-terminus of the mature protein or polypeptide. In general, the signalsequence may be a component of the vector, or it may be a part of thePRO226-, PRO257-, PRO268-, PRO290-, PRO36006-, PRO363-, PRO365-,PRO382-, PRO444-, PRO705-, PRO1071-, PRO1125-, PRO1134-, PRO1155-,PRO1281-, PRO1343-, PRO1379-, PRO1380-, PRO1387-, PRO1419-, PRO1433-,PRO1474-, PRO1550-, PRO1571-, PRO1572-, PRO1759-, PRO1904-, PRO35193-,PRO4341-, PRO4348-, PRO4369-, PRO4381-, PRO4407-, PRO4425-, PRO4985-,PRO4989-, PRO5737-, PRO5800-, PRO5993-, PRO6017-, PRO7174-, PRO9744-,PRO9821-, PRO9852-, PRO9873-, PRO10196-, PRO34778-, PRO20233-,PRO21956-, PRO57290-, PRO38465-, PRO38683- or PRO85161-encoding DNA thatis inserted into the vector. The signal sequence may be a prokaryoticsignal sequence selected, for example, from the group of the alkalinephosphatase, penicillinase, lpp, or heat-stable enterotoxin II leaders.For yeast secretion the signal sequence may be, e.g., the yeastinvertase leader, alpha factor leader (including Saccharomyces andKluyveromyces α-factor leaders, the latter described in U.S. Pat. No.5,010,182), or acid phosphatase leader, the C. albicans glucoamylaseleader (EP 362,179 published 4 Apr. 1990), or the signal described in WO90/13646 published 15 Nov. 1990. In mammalian cell expression, mammaliansignal sequences may be used to direct secretion of the protein, such assignal sequences from secreted polypeptides of the same or relatedspecies, as well as viral secretory leaders.

Both expression and cloning vectors contain a nucleic acid sequence thatenables the vector to replicate in one or more selected host cells. Suchsequences are well known for a variety of bacteria, yeast, and viruses.The origin of replication from the plasmid pBR322 is suitable for mostGram-negative bacteria, the 2μ plasmid origin is suitable for yeast, andvarious viral origins (SV40, polyoma, adenovirus, VSV or BPV) are usefulfor cloning vectors in mammalian cells.

Expression and cloning vectors will typically contain a selection gene,also termed a selectable marker. Typical selection genes encode proteinsthat (a) confer resistance to antibiotics or other toxins, e.g.,ampicillin, neomycin, methotrexate, or tetracycline, (b) complementauxotrophic deficiencies, or (c) supply critical nutrients not availablefrom complex media, e.g., the gene encoding D-alanine racemase forBacilli.

An example of suitable selectable markers for mammalian cells are thosethat enable the identification of cells competent to take up thePRO226-, PRO257-, PRO268-, PRO290-, PRO36006-, PRO363-, PRO365-,PRO382-, PRO444-, PRO705-, PRO1071-, PRO1125-, PRO1134-, PRO1155-,PRO1281-, PRO1343-, PRO1379-, PRO1380-, PRO1387-, PRO1419-, PRO1433-,PRO1474-, PRO1550-, PRO1571-, PRO1572-, PRO1759-, PRO1904-, PRO35193-,PRO4341-, PRO4348-, PRO4369-, PRO4381-, PRO4407-, PRO4425-, PRO4985-,PRO4989-, PRO5737-, PRO5800-, PRO5993-, PRO6017-, PRO7174-, PRO9744-,PRO9821-, PRO9852-, PRO9873-, PRO10196-, PRO34778-, PRO20233-,PRO21956-, PRO57290-, PRO38465-, PRO38683- or PRO85161-encoding nucleicacid, such as DHFR or thymidine kinase. An appropriate host cell whenwild-type DHFR is employed is the CHO cell line deficient in DHFRactivity, prepared and propagated as described by Urlaub et al., Proc.Natl. Acad. Sci. USA, 77:4216 (1980). A suitable selection gene for usein yeast is the trp1 gene present in the yeast plasmid YRp7 [Stinchcombet al., Nature, 282:39 (1979); Kingsman et al., Gene, 7:141 (1979);Tschemper et al., Gene, 10:157 (1980)]. The trp 1 gene provides aselection marker for a mutant strain of yeast lacking the ability togrow in tryptophan, for example, ATCC No. 44076 or PEP4-1 [Jones,Genetics, 85:12 (1977)].

Expression and cloning vectors usually contain a promoter operablylinked to the PRO226-, PRO257-, PRO268-, PRO290-, PRO36006-, PRO363-,PRO365-, PRO382-, PRO444-, PRO705-, PRO1071-, PRO1125-, PRO1134-,PRO1155-, PRO1281-, PRO1343-, PRO1379-, PRO1380-, PRO1387-, PRO1419-,PRO1433-, PRO1474-, PRO1550-, PRO1571-, PRO1572-, PRO1759-, PRO1904-,PRO35193-, PRO4341-, PRO4348-, PRO4369-, PRO4381-, PRO4407-, PRO4425-,PRO4985-, PRO4989-, PRO5737-, PRO5800-, PRO5993-, PRO6017-, PRO7174-,PRO9744-, PRO9821-, PRO9852-, PRO9873-, PRO10196-, PRO34778-, PRO20233-,PRO21956-, PRO57290-, PRO38465-, PRO38683- or PRO85161-encoding nucleicacid sequence to direct mRNA synthesis. Promoters recognized by avariety of potential host cells are well known. Promoters suitable foruse with prokaryotic hosts include the β-lactamase and lactose promotersystems [Chang et al., Nature, 275:615 (1978); Goeddel et al., Nature,281:544 (1979)], alkaline phosphatase, a tryptophan (trp) promotersystem [Goeddel, Nucleic Acids Res., 8:4057 (1980); EP 36,776], andhybrid promoters such as the tac promoter [deBoer et al., Proc. Natl.Acad. Sci. USA, 80:21-25 (1983)]. Promoters for use in bacterial systemsalso will contain a Shine-Dalgarno (S.D.) sequence operably linked tothe DNA encoding PRO226, PRO257, PRO268, PRO290, PRO36006, PRO363,PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155,PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474,PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348,PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800,PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196,PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161polypeptides.

Examples of suitable promoting sequences for use with yeast hostsinclude the promoters for 3-phosphoglycerate kinase [Hitzeman et al., J.Biol. Chem., 255:2073 (1980)] or other glycolytic enzymes [Hess et al.,J. Adv. Enzyme Reg., 7:149 (1968); Holland, Biochemistry, 17:4900(1978)], such as enolase, glyceraldehyde-3-phosphate dehydrogenase,hexokinase, pyruvate decarboxylase, phosphofructokinase,glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvatekinase, triosephosphate isomerase, phosphoglucose isomerase, andglucokinase.

Other yeast promoters, which are inducible promoters having theadditional advantage of transcription controlled by growth conditions,are the promoter regions for alcohol dehydrogenase 2, isocytochrome C,acid phosphatase, degradative enzymes associated with nitrogenmetabolism, metallothionein, glyceraldehyde-3-phosphate dehydrogenase,and enzymes responsible for maltose and galactose utilization. Suitablevectors and promoters for use in yeast expression are further describedin EP 73,657.

PRO226, PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382,PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343,PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571,PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381,PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017,PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196, PRO34778,PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161transcription from vectors in mammalian host cells is controlled, forexample, by promoters obtained from the genomes of viruses such aspolyoma virus, fowlpox virus (UK 2,211,504 published 5 Jul. 1989),adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcomavirus, cytomegalovirus, a retrovirus, hepatitis-B virus and Simian Virus40 (SV40), from heterologous mammalian promoters, e.g., the actinpromoter or an immunoglobulin promoter, and from heat-shock promoters,provided such promoters are compatible with the host cell systems.

Transcription of a DNA encoding the PRO226, PRO257, PRO268, PRO290,PRO36006, PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125,PRO1134, PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419,PRO1433, PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193,PRO4341, PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989,PRO5737, PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852,PRO9873, PRO10196, PRO34778, PRO20233, PRO21956, PRO57290, PRO38465,PRO38683 or PRO85161 polypeptide by higher eukaryotes may be increasedby inserting an enhancer sequence into the vector. Enhancers arecis-acting elements of DNA, usually about from 10 to 300 bp, that act ona promoter to increase its transcription. Many enhancer sequences arenow known from mammalian genes (globin, elastase, albumin,α-fetoprotein, and insulin). Typically, however, one will use anenhancer from a eukaryotic cell virus. Examples include the SV40enhancer on the late side of the replication origin (bp 100-270), thecytomegalovirus early promoter enhancer, the polyoma enhancer on thelate side of the replication origin, and adenovirus enhancers. Theenhancer may be spliced into the vector at a position 5′ or 3′ to thePRO226, PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382,PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343,PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571,PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381,PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017,PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196, PRO34778,PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161 codingsequence, but is preferably located at a site 5′ from the promoter.

Expression vectors used in eukaryotic host cells (yeast, fungi, insect,plant, animal, human, or nucleated cells from other multicellularorganisms) will also contain sequences necessary for the termination oftranscription and for stabilizing the mRNA. Such sequences are commonlyavailable from the 5′ and, occasionally 3′, untranslated regions ofeukaryotic or viral DNAs or cDNAs. These regions contain nucleotidesegments transcribed as polyadenylated fragments in the untranslatedportion of the mRNA encoding PRO226, PRO257, PRO268, PRO290, PRO36006,PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125, PRO1134,PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433,PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341,PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737,PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873,PRO10196, PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 orPRO85161 polypeptides.

Still other methods, vectors, and host cells suitable for adaptation tothe synthesis of PRO226, PRO257, PRO268, PRO290, PRO36006, PRO363,PRO365, PRO382, PRO444, PRO705, PRO1071, PRO125, PRO134, PRO1155,PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474,PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348,PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800,PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196,PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161polypeptides in recombinant vertebrate cell culture are described inGething et al., Nature, 293:620-625 (1981); Mantei et al., Nature,281:40-46 (1979); EP 117,060; and EP 117,058.

4. Detecting Gene Amplification/Expression

Gene amplification and/or expression may be measured in a sampledirectly, for example, by conventional Southern blotting, Northernblotting to quantitate the transcription of mRNA [Thomas, Proc. Natl.Acad. Sci. USA, 77:5201-5205 (1980)], dot blotting (DNA analysis), or insitu hybridization, using an appropriately labeled probe, based on thesequences provided herein. Alternatively, antibodies may be employedthat can recognize specific duplexes, including DNA duplexes, RNAduplexes, and DNA-RNA hybrid duplexes or DNA-protein duplexes. Theantibodies in turn may be labeled and the assay may be carried out wherethe duplex is bound to a surface, so that upon the formation of duplexon the surface, the presence of antibody bound to the duplex can bedetected.

Gene expression, alternatively, may be measured by immunologicalmethods, such as immunohistochemical staining of cells or tissuesections and assay of cell culture or body fluids, to quantitatedirectly the expression of gene product. Antibodies useful forimmunohistochemical staining and/or assay of sample fluids may be eithermonoclonal or polyclonal, and may be prepared in any mammal.Conveniently, the antibodies may be prepared against a native sequencePRO226, PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382,PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343,PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571,PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381,PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017,PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196, PRO34778,PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161 polypeptideor against a synthetic peptide based on the DNA sequences providedherein or against exogenous sequence fused to PRO226, PRO257, PRO268,PRO290, PRO36006, PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071,PRO1125, PRO1134, PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387,PRO1419, PRO1433, PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904,PRO35193, PRO4341, PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985,PRO4989, PRO5737, PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821,PRO9852, PRO9873, PRO10196, PRO34778, PRO20233, PRO21956, PRO57290,PRO38465, PRO38683 or PRO85161 DNA and encoding a specific antibodyepitope.

5. Purification of Polypeptide

Forms of PRO226, PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365,PRO382, PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281,PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550,PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369,PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993,PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196,PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161polypeptides may be recovered from culture medium or from host celllysates. If membrane-bound, it can be released from the membrane using asuitable detergent solution (e.g. Triton-X 100) or by enzymaticcleavage. Cells employed in expression of PRO226, PRO257, PRO268,PRO290, PRO36006, PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071,PRO1125, PRO1134, PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387,PRO1419, PRO1433, PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904,PRO35193, PRO4341, PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985,PRO4989, PRO5737, PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821,PRO9852, PRO9873, PRO10196, PRO34778, PRO20233, PRO21956, PRO57290,PRO38465, PRO38683 or PRO85161 polypeptides can be disrupted by variousphysical or chemical means, such as freeze-thaw cycling, sonication,mechanical disruption, or cell lysing agents.

It may be desired to purify PRO226, PRO257, PRO268, PRO290, PRO36006,PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125, PRO1134,PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433,PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341,PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737,PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873,PRO10196, PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 orPRO85161 polypeptides from recombinant cell proteins or polypeptides.The following procedures are exemplary of suitable purificationprocedures: by fractionation on an ion-exchange column; ethanolprecipitation; reverse phase HPLC; chromatography on silica or on acation-exchange resin such as DEAE; chromatofocusing; SDS-PAGE; ammoniumsulfate precipitation; gel filtration using, for example, Sephadex G-75;protein A Sepharose columns to remove contaminants such as IgG; andmetal chelating columns to bind epitope-tagged forms of the PRO226,PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382, PRO444,PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343, PRO1379,PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571, PRO1572,PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381, PRO4407,PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017, PRO7174,PRO9744, PRO9821, PRO9852, PRO9873, PRO10196, PRO34778, PRO20233,PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161 polypeptide. Variousmethods of protein purification may be employed and such methods areknown in the art and described for example in Deutscher, Methods inEnzymology, 182 (1990); Scopes, Protein Purification: Principles andPractice, Springer-Verlag, New York (1982). The purification step(s)selected will depend, for example, on the nature of the productionprocess used and the particular PRO226, PRO257, PRO268, PRO290,PRO36006, PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125,PRO1134, PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419,PRO1433, PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193,PRO4341, PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989,PRO5737, PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852,PRO9873, PRO10196, PRO34778, PRO20233, PRO21956, PRO57290, PRO38465,PRO38683 or PRO85161 polypeptide produced.

E. Uses for PRO226, PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365,PRO382, PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281,PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550,PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369,PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993,PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196,PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161Polypeptides

Nucleotide sequences (or their complement) encoding PRO226, PRO257,PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382, PRO444, PRO705,PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343, PRO1379, PRO1380,PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571, PRO1572, PRO1759,PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381, PRO4407, PRO4425,PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017, PRO7174, PRO9744,PRO9821, PRO9852, PRO9873, PRO10196, PRO34778, PRO20233, PRO21956,PRO57290, PRO38465, PRO38683 or PRO85161 polypeptides have variousapplications in the art of molecular biology, including uses ashybridization probes, in chromosome and gene mapping and in thegeneration of anti-sense RNA and DNA. PRO226, PRO257, PRO268, PRO290,PRO36006, PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125,PRO1134, PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419,PRO1433, PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193,PRO4341, PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989,PRO5737, PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852,PRO9873, PRO10196, PRO34778, PRO20233, PRO21956, PRO57290, PRO38465,PRO38683 or PRO85161 nucleic acid will also be useful for thepreparation of PRO226, PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365,PRO382, PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281,PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550,PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369,PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993,PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196,PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161polypeptides by the recombinant techniques described herein.

The full-length native sequence PRO226, PRO257, PRO268, PRO290,PRO36006, PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125,PRO1134, PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419,PRO1433, PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193,PRO4341, PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989,PRO5737, PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852,PRO9873, PRO10196, PRO34778, PRO20233, PRO21956, PRO57290, PRO38465,PRO38683 or PRO85161 gene, or portions thereof, may be used ashybridization probes for a cDNA library to isolate the full-lengthPRO226, PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382,PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343,PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571,PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381,PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017,PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196, PRO34778,PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161 cDNA or toisolate still other cDNAs (for instance, those encodingnaturally-occurring variants of PRO226, PRO257, PRO268, PRO290,PRO36006, PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125,PRO1134, PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419,PRO1433, PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193,PRO4341, PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989,PRO5737, PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852,PRO9873, PRO10196, PRO34778, PRO20233, PRO21956, PRO57290, PRO38465,PRO38683 or PRO85161 polypeptides or PRO226, PRO257, PRO268, PRO290,PRO36006, PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125,PRO1134, PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419,PRO1433, PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193,PRO4341, PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989,PRO5737, PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852,PRO9873, PRO10196, PRO34778, PRO20233, PRO21956, PRO57290, PRO38465,PRO38683 or PRO85161 polypeptides from other species) which have adesired sequence identity to the native PRO226, PRO257, PRO268, PRO290,PRO36006, PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125,PRO1134, PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419,PRO1433, PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193,PRO4341, PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989,PRO5737, PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852,PRO9873, PRO10196, PRO34778, PRO20233, PRO21956, PRO57290, PRO38465,PRO38683 or PRO85161 sequence disclosed herein. Optionally, the lengthof the probes will be about 20 to about 50 bases. The hybridizationprobes may be derived from at least partially novel regions of the fulllength native nucleotide sequence wherein those regions may bedetermined without undue experimentation or from genomic sequencesincluding promoters, enhancer elements and introns of native sequencePRO226, PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382,PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343,PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571,PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381,PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017,PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196, PRO34778,PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161. By way ofexample, a screening method will comprise isolating the coding region ofthe PRO226, PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382,PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343,PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571,PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381,PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017,PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196, PRO34778,PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161 gene usingthe known DNA sequence to synthesize a selected probe of about 40 bases.Hybridization probes may be labeled by a variety of labels, includingradionucleotides such as ³²P or ³⁵S, or enzymatic labels such asalkaline phosphatase coupled to the probe via avidin/biotin couplingsystems. Labeled probes having a sequence complementary to that of thePRO226, PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382,PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343,PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571,PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381,PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017,PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196, PRO34778,PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161 gene of thepresent invention can be used to screen libraries of human cDNA, genomicDNA or mRNA to determine which members of such libraries the probehybridizes to. Hybridization techniques are described in further detailin the Examples below.

Any EST sequences disclosed in the present application may similarly beemployed as probes, using the methods disclosed herein.

Other useful fragments of the PRO226, PRO257, PRO268, PRO290, PRO36006,PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125, PRO1134,PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433,PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341,PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737,PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873,PRO10196, PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 orPRO85161 nucleic acids include antisense or sense oligonucleotidescomprising a singe-stranded nucleic acid sequence (either RNA or DNA)capable of binding to target PRO226, PRO257, PRO268, PRO290, PRO36006,PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125, PRO1134,PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433,PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341,PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737,PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873,PRO10196, PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 orPRO85161 mRNA (sense) or PRO226, PRO257, PRO268, PRO290, PRO36006,PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125, PRO1134,PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433,PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341,PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737,PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873,PRO10196, PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 orPRO85161 DNA (antisense) sequences. Antisense or sense oligonucleotides,according to the present invention, comprise a fragment of the codingregion of PRO226, PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365,PRO382, PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281,PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550,PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369,PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993,PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196,PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161DNA. Such a fragment generally comprises at least about 14 nucleotides,preferably from about 14 to 30 nucleotides. The ability to derive anantisense or a sense oligonucleotide, based upon a cDNA sequenceencoding a given protein is described in, for example, Stein and Cohen(Cancer Res. 48:2659, 1988) and van der Krol et al. (BioTechniques6:958, 1988).

Binding of antisense or sense oligonucleotides to target nucleic acidsequences results in the formation of duplexes that block transcriptionor translation of the target sequence by one of several means, includingenhanced degradation of the duplexes, premature termination oftranscription or translation, or by other means. The antisenseoligonucleotides thus may be used to block expression of PRO226, PRO257,PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382, PRO444, PRO705,PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343, PRO1379, PRO1380,PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571, PRO1572, PRO1759,PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381, PRO4407, PRO4425,PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017, PRO7174, PRO9744,PRO9821, PRO9852, PRO9873, PRO10196, PRO34778, PRO20233, PRO21956,PRO57290, PRO38465, PRO38683 or PRO85161. Antisense or senseoligonucleotides further comprise oligonucleotides having modifiedsugar-phosphodiester backbones (or other sugar linkages, such as thosedescribed in WO 91/06629) and wherein such sugar linkages are resistantto endogenous nucleases. Such oligonucleotides with resistant sugarlinkages are stable in vivo (i.e., capable of resisting enzymaticdegradation) but retain sequence specificity to be able to bind totarget nucleotide sequences.

Other examples of sense or antisense oligonucleotides include thoseoligonucleotides which are covalently linked to organic moieties, suchas those described in WO 90/10048, and other moieties that increasesaffinity of the oligonucleotide for a target nucleic acid sequence, suchas poly-(L-lysine). Further still, intercalating agents, such asellipticine, and alkylating agents or metal complexes may be attached tosense or antisense oligonucleotides to modify binding specificities ofthe antisense or sense oligonucleotide for the target nucleotidesequence.

Antisense or sense oligonucleotides may be introduced into a cellcontaining the target nucleic acid sequence by any gene transfer method,including, for example, CaPO₄-mediated DNA transfection,electroporation, or by using gene transfer vectors such as Epstein-Barrvirus. In a preferred procedure, an antisense or sense oligonucleotideis inserted into a suitable retroviral vector. A cell containing thetarget nucleic acid sequence is contacted with the recombinantretroviral vector, either in vivo or ex vivo. Suitable retroviralvectors include, but are not limited to, those derived from the murineretrovirus M-MuLV, N2 (a retrovirus derived from M-MuLV), or the doublecopy vectors designated DCT5A, DCT5B and DCT5C (see WO 90/13641).

Sense or antisense oligonucleotides also may be introduced into a cellcontaining the target nucleotide sequence by formation of a conjugatewith a ligand binding molecule, as described in WO 91/04753. Suitableligand binding molecules include, but are not limited to, cell surfacereceptors, growth factors, other cytokines, or other ligands that bindto cell surface receptors. Preferably, conjugation of the ligand bindingmolecule does not substantially interfere with the ability of the ligandbinding molecule to bind to its corresponding molecule or receptor, orblock entry of the sense or antisense oligonucleotide or its conjugatedversion into the cell.

Alternatively, a sense or an antisense oligonucleotide may be introducedinto a cell containing the target nucleic acid sequence by formation ofan oligonucleotide-lipid complex, as described in WO 90/10448. The senseor antisense oligonucleotide-lipid complex is preferably dissociatedwithin the cell by an endogenous lipase.

Antisense or sense RNA or DNA molecules are generally at least about 5bases in length, about 10 bases in length, about 15 bases in length,about 20 bases in length, about 25 bases in length, about 30 bases inlength, about 35 bases in length, about 40 bases in length, about 45bases in length, about 50 bases in length, about 55 bases in length,about 60 bases in length, about 65 bases in length, about 70 bases inlength, about 75 bases in length, about 80 bases in length, about 85bases in length, about 90 bases in length, about 95 bases in length,about 100 bases in length, or more.

The probes may also be employed in PCR techniques to generate a pool ofsequences for identification of closely related PRO226, PRO257, PRO268,PRO290, PRO36006, PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071,PRO1125, PRO1134, PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387,PRO1419, PRO1433, PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904,PRO35193, PRO4341, PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985,PRO4989, PRO5737, PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821,PRO9852, PRO9873, PRO10196, PRO34778, PRO20233, PRO21956, PRO57290,PRO38465, PRO38683 or PRO85161 coding sequences.

Nucleotide sequences encoding a PRO226, PRO257, PRO268, PRO290,PRO36006, PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125,PRO1134, PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419,PRO1433, PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193,PRO4341, PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989,PRO5737, PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852,PRO9873, PRO10196, PRO34778, PRO20233, PRO21956, PRO57290, PRO38465,PRO38683 or PRO85161 polypeptide can also be used to constructhybridization probes for mapping the gene which encodes that PRO226,PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382, PRO444,PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343, PRO1379,PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571, PRO1572,PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381, PRO4407,PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017, PRO7174,PRO9744, PRO9821, PRO9852, PRO9873, PRO10196, PRO34778, PRO20233,PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161 polypeptide and forthe genetic analysis of individuals with genetic disorders. Thenucleotide sequences provided herein may be mapped to a chromosome andspecific regions of a chromosome using known techniques, such as in situhybridization, linkage analysis against known chromosomal markers, andhybridization screening with libraries.

When the coding sequences for PRO226, PRO257, PRO268, PRO290, PRO36006,PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125, PRO1134,PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433,PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341,PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737,PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873,PRO10196, PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 orPRO85161 encode a protein which binds to another protein (for example,where the PRO226, PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365,PRO382, PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281,PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550,PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369,PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993,PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196,PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161is a receptor), the PRO226, PRO257, PRO268, PRO290, PRO36006, PRO363,PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125, PRO134, PRO1155,PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474,PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348,PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800,PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196,PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161polypeptide can be used in assays to identify the other proteins ormolecules involved in the binding interaction. By such methods,inhibitors of the receptor/ligand binding interaction can be identified.Proteins involved in such binding interactions can also be used toscreen for peptide or small molecule inhibitors or agonists of thebinding interaction. Also, the receptor PRO226, PRO257, PRO268, PRO290,PRO36006, PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125,PRO1134, PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419,PRO1433, PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193,PRO4341, PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989,PRO5737, PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852,PRO9873, PRO10196, PRO34778, PRO20233, PRO21956, PRO57290, PRO38465,PRO38683 or PRO85161 can be used to isolate correlative ligand(s).Screening assays can be designed to find lead compounds that mimic thebiological activity of a native PRO226, PRO257, PRO268, PRO290,PRO36006, PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125,PRO1134, PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419,PRO1433, PRO1474, PRO550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193,PRO4341, PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989,PRO5737, PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852,PRO9873, PRO10196, PRO34778, PRO20233, PRO21956, PRO57290, PRO38465,PRO38683 or PRO85161 polypeptide or a receptor for PRO226, PRO257,PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382, PRO444, PRO705,PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343, PRO1379, PRO1380,PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571, PRO1572, PRO1759,PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381, PRO4407, PRO4425,PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017, PRO7174, PRO9744,PRO9821, PRO9852, PRO9873, PRO10196, PRO34778, PRO20233, PRO21956,PRO57290, PRO38465, PRO38683 or PRO85161 polypeptides. Such screeningassays will include assays amenable to high-throughput screening ofchemical libraries, making them particularly suitable for identifyingsmall molecule drug candidates. Small molecules contemplated includesynthetic organic or inorganic compounds. The assays can be performed ina variety of formats, including protein-protein binding assays,biochemical screening assays, immunoassays and cell based assays, whichare well characterized in the art.

Nucleic acids which encode PRO226, PRO257, PRO268, PRO290, PRO36006,PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125, PRO1134,PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433,PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341,PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737,PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873,PRO10196, PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 orPRO85161 polypeptides or its modified forms can also be used to generateeither transgenic animals or “knock out” animals which, in turn, areuseful in the development and screening of therapeutically usefulreagents. A transgenic animal (e.g., a mouse or rat) is an animal havingcells that contain a transgene, which transgene was introduced into theanimal or an ancestor of the animal at a prenatal, e.g., an embryonicstage. A transgene is a DNA which is integrated into the genome of acell from which a transgenic animal develops. The invention providescDNA encoding a PRO226, PRO257, PRO268, PRO290, PRO36006, PRO363,PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155,PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474,PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348,PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800,PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196,PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161polypeptide which can be used to clone genomic DNA encoding a PRO226,PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382, PRO444,PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343, PRO1379,PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571, PRO1572,PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381, PRO4407,PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017, PRO7174,PRO9744, PRO9821, PRO9852, PRO9873, PRO10196, PRO34778, PRO20233,PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161 polypeptide inaccordance with established techniques and the genomic sequences used togenerate transgenic animals that contain cells which express DNAencoding PRO226, PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365,PRO382, PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281,PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550,PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369,PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993,PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196,PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161polypeptides. Any technique known in the art may be used to introduce atarget gene transgene into animals to produce the founder lines oftransgenic animals. Such techniques include, but are not limited topronuclear microinjection (U.S. Pat. Nos. 4,873,191, 4,736,866 and4,870,009); retrovirus mediated gene transfer into germ lines (Van derPutten, et al., Proc. Natl. Acad. Sci. USA, 82:6148-6152 (1985)); genetargeting in embryonic stem cells (Thompson, et al., Cell, 56:313-321(1989)); nonspecific insertional inactivation using a gene trap vector(U.S. Pat. No. 6,436,707); electroporation of embryos (Lo, Mol. Cell.Biol., 3:1803-1814 (1983)); and sperm-mediated gene transfer (Lavitrano,et al., Cell, 57:717-723 (1989)); etc. Typically, particular cells wouldbe targeted for a PRO226, PRO257, PRO268, PRO290, PRO36006, PRO363,PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155,PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474,PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348,PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800,PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196,PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161transgene incorporation with tissue-specific enhancers. Transgenicanimals that include a copy of a transgene encoding a PRO226, PRO257,PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382, PRO444, PRO705,PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343, PRO1379, PRO1380,PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571, PRO1572, PRO1759,PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381, PRO4407, PRO4425,PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017, PRO7174, PRO9744,PRO9821, PRO9852, PRO9873, PRO10196, PRO34778, PRO20233, PRO21956,PRO57290, PRO38465, PRO38683 or PRO85161 polypeptide introduced into thegerm line of the animal at an embryonic stage can be used to examine theeffect of increased expression of DNA encoding PRO226, PRO257, PRO268,PRO290, PRO36006, PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071,PRO1125, PRO1134, PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387,PRO1419, PRO1433, PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904,PRO35193, PRO4341, PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985,PRO4989, PRO5737, PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821,PRO9852, PRO9873, PRO10196, PRO34778, PRO20233, PRO21956, PRO57290,PRO38465, PRO38683 or PRO85161 polypeptides. Such animals can be used astester animals for reagents thought to confer protection from, forexample, pathological conditions associated with its overexpression. Inaccordance with this facet of the invention, an animal is treated withthe reagent and a reduced incidence of the pathological condition,compared to untreated animals bearing the transgene, would indicate apotential therapeutic intervention for the pathological condition.Alternatively, non-human homologues of PRO226, PRO257, PRO268, PRO290,PRO36006, PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125,PRO1134, PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419,PRO1433, PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193,PRO4341, PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989,PRO5737, PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852,PRO9873, PRO10196, PRO34778, PRO20233, PRO21956, PRO57290, PRO38465,PRO38683 or PRO85161 polypeptides can be used to construct a PRO226,PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382, PRO444,PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343, PRO1379,PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571, PRO1572,PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381, PRO4407,PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017, PRO7174,PRO9744, PRO9821, PRO9852, PRO9873, PRO10196, PRO34778, PRO20233,PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161 “knock out” animalwhich has a defective or altered gene encoding PRO226, PRO257, PRO268,PRO290, PRO36006, PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071,PRO1125, PRO1134, PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387,PRO1419, PRO1433, PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904,PRO35193, PRO4341, PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985,PRO4989, PRO5737, PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821,PRO9852, PRO9873, PRO10196, PRO34778, PRO20233, PRO21956, PRO57290,PRO38465, PRO38683 or PRO85161 proteins as a result of homologousrecombination between the endogenous gene encoding PRO226, PRO257,PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382, PRO444, PRO705,PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343, PRO1379, PRO1380,PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571, PRO1572, PRO1759,PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381, PRO4407, PRO4425,PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017, PRO7174, PRO9744,PRO9821, PRO9852, PRO9873, PRO10196, PRO34778, PRO20233, PRO21956,PRO57290, PRO38465, PRO38683 or PRO85161 polypeptides and alteredgenomic DNA encoding PRO226, PRO257, PRO268, PRO290, PRO36006, PRO363,PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155,PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474,PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348,PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800,PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196,PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161polypeptides introduced into an embryonic stem cell of the animal.Preferably the knock out animal is a mammal. More preferably, the mammalis a rodent such as a rat or mouse. For example, cDNA encoding PRO226,PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382, PRO444,PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343, PRO1379,PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571, PRO1572,PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381, PRO4407,PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017, PRO7174,PRO9744, PRO9821, PRO9852, PRO9873, PRO10196, PRO34778, PRO20233,PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161 polypeptides can beused to clone genomic DNA encoding PRO226, PRO257, PRO268, PRO290,PRO36006, PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125,PRO1134, PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419,PRO1433, PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193,PRO4341, PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989,PRO5737, PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852,PRO9873, PRO10196, PRO34778, PRO20233, PRO21956, PRO57290, PRO38465,PRO38683 or PRO85161 polypeptides in accordance with establishedtechniques. A portion of the genomic DNA encoding the PRO226, PRO257,PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382, PRO444, PRO705,PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343, PRO1379, PRO1380,PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571, PRO1572, PRO1759,PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381, PRO4407, PRO4425,PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017, PRO7174, PRO9744,PRO9821, PRO9852, PRO9873, PRO10196, PRO34778, PRO20233, PRO21956,PRO57290, PRO38465, PRO38683 or PRO85161 polypeptide can be deleted orreplaced with another gene, such as a gene encoding a selectable markerwhich can be used to monitor integration. Typically, several kilobasesof unaltered flanking DNA (both at the 5′ and 3′ ends) are included inthe vector [see e.g., Thomas and Capecchi, Cell, 51:503 (1987) for adescription of homologous recombination vectors]. The vector isintroduced into an embryonic stem cell line (e.g., by electroporation)and cells in which the introduced DNA has homologously recombined withthe endogenous DNA are selected [see e.g., Li et al., Cell, 69:915(1992)]. The selected cells are then injected into a blastocyst of ananimal (e.g., a mouse or rat) to form aggregation chimeras [see e.g.,Bradley, in Teratocarcinomas and Embryonic Stem Cells: A PracticalApproach, E. J. Robertson, ed. (IRL, Oxford, 1987), pp. 113-152]. Achimeric embryo can then be implanted into a suitable pseudopregnantfemale foster animal and the embryo brought to term to create a “knockout” animal. Progeny harboring the homologously recombined DNA in theirgerm cells can be identified by standard techniques and used to breedanimals in which all cells of the animal contain the homologouslyrecombined DNA. Knockout animals can be characterized for instance, fortheir ability to defend against certain pathological conditions and fortheir development of pathological conditions due to absence of the geneencoding the PRO226, PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365,PRO382, PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281,PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550,PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369,PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993,PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196,PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161polypeptide.

In addition, knockout mice can be highly informative in the discovery ofgene function and pharmaceutical utility for a drug target, as well asin the determination of the potential on-target side effects associatedwith a given target. Gene function and physiology are so well conservedbetween mice and humans, since they are both mammals and contain similarnumbers of genes, which are highly conserved between the species. It hasrecently been well documented, for example, that 98% of genes on mousechromosome 16 have a human ortholog (Mural et al., Science 296:1661-71(2002)).

Although gene targeting in embryonic stem (ES) cells has enabled theconstruction of mice with null mutations in many genes associated withhuman disease, not all genetic diseases are attributable to nullmutations. One can design valuable mouse models of human diseases byestablishing a method for gene replacement (knock-in) which will disruptthe mouse locus and introduce a human counterpart with mutation,Subsequently one can conduct in vivo drug studies targeting the humanprotein (Kitamoto et. Al., Biochemical and Biophysical Res. Commun.,222:742-47 (1996)).

Nucleic acid encoding the PRO226, PRO257, PRO268, PRO290, PRO36006,PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125, PRO1134,PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433,PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341,PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737,PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873,PRO10196, PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 orPRO85161 polypeptides may also be used in gene therapy. In gene therapyapplications, genes are introduced into cells in order to achieve invivo synthesis of a therapeutically effective genetic product, forexample for replacement of a defective gene. “Gene therapy” includesboth conventional gene therapy where a lasting effect is achieved by asingle treatment, and the administration of gene therapeutic agents,which involves the one time or repeated administration of atherapeutically effective DNA or mRNA. Antisense RNAs and DNAs can beused as therapeutic agents for blocking the expression of certain genesin vivo. It has already been shown that short antisense oligonucleotidescan be imported into cells where they act as inhibitors, despite theirlow intracellular concentrations caused by their restricted uptake bythe cell membrane. (Zamecnik et al., Proc. Natl. Acad. Sci. USA83:4143-4146 [1986]). The oligonucleotides can be modified to enhancetheir uptake, e.g. by substituting their negatively chargedphosphodiester groups by uncharged groups.

There are a variety of techniques available for introducing nucleicacids into viable cells. The techniques vary depending upon whether thenucleic acid is transferred into cultured cells in vitro, or in vivo inthe cells of the intended host. Techniques suitable for the transfer ofnucleic acid into mammalian cells in vitro include the use of liposomes,electroporation, microinjection, cell fusion, DEAE-dextran, the calciumphosphate precipitation method, etc. The currently preferred in vivogene transfer techniques include transfection with viral (typicallyretroviral) vectors and viral coat protein-liposome mediatedtransfection (Dzau et al., Trends in Biotechnology 11, 205-210 [1993]).In some situations it is desirable to provide the nucleic acid sourcewith an agent that targets the target cells, such as an antibodyspecific for a cell surface membrane protein or the target cell, aligand for a receptor on the target cell, etc. Where liposomes areemployed, proteins which bind to a cell surface membrane proteinassociated with endocytosis may be used for targeting and/or tofacilitate uptake, e.g. capsid proteins or fragments thereof tropic fora particular cell type, antibodies for proteins which undergointernalization in cycling, proteins that target intracellularlocalization and enhance intracellular half-life. The technique ofreceptor-mediated endocytosis is described, for example, by Wu et al.,J. Biol. Chem. 262, 4429-4432 (1987); and Wagner et al., Proc. Natl.Acad. Sci. USA 87, 3410-3414 (1990). For review of gene marking and genetherapy protocols see Anderson et al., Science 256, 808-813 (1992).

The PRO226, PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382,PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343,PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571,PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381,PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017,PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196, PRO34778,PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161polypeptides described herein may also be employed as molecular weightmarkers for protein electrophoresis purposes and the isolated nucleicacid sequences may be used for recombinantly expressing those markers.

The nucleic acid molecules encoding the PRO226, PRO257, PRO268, PRO290,PRO36006, PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125,PRO1134, PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419,PRO1433, PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193,PRO4341, PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989,PRO5737, PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852,PRO9873, PRO10196, PRO34778, PRO20233, PRO21956, PRO57290, PRO38465,PRO38683 or PRO85161 polypeptides or fragments thereof described hereinare useful for chromosome identification. In this regard, there existsan ongoing need to identify new chromosome markers, since relatively fewchromosome marking reagents, based upon actual sequence data arepresently available. Each PRO226, PRO257, PRO268, PRO290, PRO36006,PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125, PRO1134,PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433,PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341,PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737,PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873,PRO10196, PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 orPRO85161 nucleic acid molecule of the present invention can be used as achromosome marker.

The PRO226, PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382,PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343,PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571,PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381,PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017,PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196, PRO34778,PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161polypeptides and nucleic acid molecules of the present invention mayalso be used diagnostically for tissue typing, wherein the PRO226,PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382, PRO444,PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343, PRO1379,PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571, PRO1572,PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381, PRO4407,PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017, PRO7174,PRO9744, PRO9821, PRO9852, PRO9873, PRO10196, PRO34778, PRO20233,PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161 polypeptides of thepresent invention may be differentially expressed in one tissue ascompared to another, preferably in a diseased tissue as compared to anormal tissue of the same tissue type. PRO226, PRO257, PRO268, PRO290,PRO36006, PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125,PRO1134, PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419,PRO1433, PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193,PRO4341, PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989,PRO5737, PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852,PRO9873, PRO10196, PRO34778, PRO20233, PRO21956, PRO57290, PRO38465,PRO38683 or PRO85161 nucleic acid molecules will find use for generatingprobes for PCR, Northern analysis, Southern analysis and Westernanalysis.

The PRO226, PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382,PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343,PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571,PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381,PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017,PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196, PRO34778,PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161polypeptides described herein may also be employed as therapeuticagents. The PRO226, PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365,PRO382, PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281,PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550,PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369,PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993,PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196,PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161polypeptides of the present invention can be formulated according toknown methods to prepare pharmaceutically useful compositions, wherebythe PRO226, PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382,PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343,PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571,PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381,PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017,PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196, PRO34778,PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161 producthereof is combined in admixture with a pharmaceutically acceptablecarrier vehicle. Therapeutic formulations are prepared for storage bymixing the active ingredient having the desired degree of purity withoptional physiologically acceptable carriers, excipients or stabilizers(Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)),in the form of lyophilized formulations or aqueous solutions. Acceptablecarriers, excipients or stabilizers are nontoxic to recipients at thedosages and concentrations employed, and include buffers such asphosphate, citrate and other organic acids; antioxidants includingascorbic acid; low molecular weight (less than about 10 residues)polypeptides; proteins, such as serum albumin, gelatin orimmunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone,amino acids such as glycine, glutamine, asparagine, arginine or lysine;monosaccharides, disaccharides and other carbohydrates includingglucose, mannose, or dextrins; chelating agents such as EDTA; sugaralcohols such as mannitol or sorbitol; salt-forming counterions such assodium; and/or nonionic surfactants such as TWEEN™, PLURONICS™ or PEG.

The formulations to be used for in vivo administration must be sterile.This is readily accomplished by filtration through sterile filtrationmembranes, prior to or following lyophilization and reconstitution.

Therapeutic compositions herein generally are placed into a containerhaving a sterile access port, for example, an intravenous solution bagor vial having a stopper pierceable by a hypodermic injection needle.

The route of administration is in accord with known methods, e.g.injection or infusion by intravenous, intraperitoneal, intracerebral,intramuscular, intraocular, intraarterial or intralesional routes,topical administration, or by sustained release systems.

Dosages and desired drug concentrations of pharmaceutical compositionsof the present invention may vary depending on the particular useenvisioned. The determination of the appropriate dosage or route ofadministration is well within the skill of an ordinary physician. Animalexperiments provide reliable guidance for the determination of effectivedoses for human therapy. Interspecies scaling of effective doses can beperformed following the principles laid down by Mordenti, J. andChappell, W. “The use of interspecies scaling in toxicokinetics” InToxicokinetics and New Drug Development, Yacobi et al., Eds., PergamonPress, New York 1989, pp. 42-96.

When in vivo administration of a PRO226, PRO257, PRO268, PRO290,PRO36006, PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125,PRO1134, PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419,PRO1433, PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193,PRO4341, PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989,PRO5737, PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852,PRO9873, PRO10196, PRO34778, PRO20233, PRO21956, PRO57290, PRO38465,PRO38683 or PRO85161 polypeptide or agonist or antagonist thereof isemployed, normal dosage amounts may vary from about 10 ng/kg to up to100 mg/kg of mammal body weight or more per day, preferably about 1μg/kg/day to 10 mg/kg/day, depending upon the route of administration.Guidance as to particular dosages and methods of delivery is provided inthe literature; see, for example, U.S. Pat. No. 4,657,760; 5,206,344; or5,225,212. It is anticipated that different formulations will beeffective for different treatment compounds and different disorders,that administration targeting one organ or tissue, for example, maynecessitate delivery in a manner different from that to another organ ortissue.

Where sustained-release administration of a PRO226, PRO257, PRO268,PRO290, PRO36006, PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071,PRO1125, PRO1134, PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387,PRO1419, PRO1433, PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904,PRO35193, PRO4341, PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985,PRO4989, PRO5737, PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821,PRO9852, PRO9873, PRO10196, PRO34778, PRO20233, PRO21956, PRO57290,PRO38465, PRO38683 or PRO85161 polypeptide is desired in a formulationwith release characteristics suitable for the treatment of any diseaseor disorder requiring administration of the PRO226, PRO257, PRO268,PRO290, PRO36006, PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071,PRO1125, PRO1134, PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387,PRO1419, PRO1433, PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904,PRO35193, PRO4341, PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985,PRO4989, PRO5737, PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821,PRO9852, PRO9873, PRO10196, PRO34778, PRO20233, PRO21956, PRO57290,PRO38465, PRO38683 or PRO85161 polypeptide, microencapsulation of thePRO226, PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382,PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343,PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571,PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381,PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017,PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196, PRO34778,PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161 polypeptideis contemplated. Microencapsulation of recombinant proteins forsustained release has been successfully performed with human growthhormone (rhGH), interferon-(rhIFN-), interleukin-2, and MN rgp120.Johnson et al., Nat. Med., 2:795-799 (1996); Yasuda, Biomed. Ther.,27:1221-1223 (1993); Hora et al., Bio/Technology, 8:755-758 (1990);Cleland, “Design and Production of Single Immunization Vaccines UsingPolylactide Polyglycolide Microsphere Systems,” in Vaccine Design: TheSubunit and Adjuvant Approach, Powell and Newman, eds, (Plenum Press:New York, 1995), pp. 439-462; WO 97/03692, WO 96/40072, WO 96/07399; andU.S. Pat. No. 5,654,010.

The sustained-release formulations of these proteins were developedusing poly-lactic-coglycolic acid (PLGA) polymer due to itsbiocompatibility and wide range of biodegradable properties. Thedegradation products of PLGA, lactic and glycolic acids, can be clearedquickly within the human body. Moreover, the degradability of thispolymer can be adjusted from months to years depending on its molecularweight and composition. Lewis, “Controlled release of bioactive agentsfrom lactide/glycolide polymer,” in: M. Chasin and R. Langer (Eds.),Biodegradable Polymers as Drug Delivery Systems (Marcel Dekker: NewYork, 1990), pp. 1-41.

This invention encompasses methods of screening compounds to identifythose that mimic the PRO226, PRO257, PRO268, PRO290, PRO36006, PRO363,PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155,PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474,PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348,PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800,PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196,PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161polypeptide (agonists) or prevent the effect of the PRO226, PRO257,PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382, PRO444, PRO705,PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343, PRO1379, PRO1380,PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571, PRO1572, PRO1759,PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381, PRO4407, PRO4425,PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017, PRO7174, PRO9744,PRO9821, PRO9852, PRO9873, PRO10196, PRO34778, PRO20233, PRO21956,PRO57290, PRO38465, PRO38683 or PRO85161 polypeptide (antagonists).Agonists that mimic a PRO226, PRO257, PRO268, PRO290, PRO36006, PRO363,PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155,PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474,PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348,PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800,PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196,PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161polypeptide would be especially valuable therapeutically in thoseinstances where a negative phenotype is observed based on findings withthe non-human transgenic animal whose genome comprises a disruption ofthe gene which encodes for the PRO226, PRO257, PRO268, PRO290, PRO36006,PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125, PRO1134,PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433,PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341,PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737,PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873,PRO10196, PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 orPRO85161 polypeptide. Antagonists that prevent the effects of a PRO226,PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382, PRO444,PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343, PRO1379,PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571, PRO1572,PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381, PRO4407,PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017, PRO7174,PRO9744, PRO9821, PRO9852, PRO9873, PRO10196, PRO34778, PRO20233,PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161 polypeptide would beespecially valuable therapeutically in those instances where a positivephenotype is observed based upon observations with the non-humantransgenic knockout animal. Screening assays for antagonist drugcandidates are designed to identify compounds that bind or complex withthe PRO226, PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382,PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343,PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571,PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381,PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017,PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196, PRO34778,PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161 polypeptideencoded by the genes identified herein, or otherwise interfere with theinteraction of the encoded polypeptide with other cellular proteins.Such screening assays will include assays amenable to high-throughputscreening of chemical libraries, making them particularly suitable foridentifying small molecule drug candidates.

The assays can be performed in a variety of formats, includingprotein-protein binding assays, biochemical screening assays,immunoassays, and cell-based assays, which are well characterized in theart.

All assays for antagonists are common in that they call for contactingthe drug candidate with a PRO226, PRO257, PRO268, PRO290, PRO36006,PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125, PRO1134,PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433,PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341,PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737,PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873,PRO10196, PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 orPRO85161 polypeptide encoded by a nucleic acid identified herein underconditions and for a time sufficient to allow these two components tointeract.

In binding assays, the interaction is binding and the complex formed canbe isolated or detected in the reaction mixture. The PRO226, PRO257,PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382, PRO444, PRO705,PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343, PRO1379, PRO1380,PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571, PRO1572, PRO1759,PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381, PRO4407, PRO4425,PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017, PRO7174, PRO9744,PRO9821, PRO9852, PRO9873, PRO10196, PRO34778, PRO20233, PRO21956,PRO57290, PRO38465, PRO38683 or PRO85161 polypeptide encoded by the geneidentified herein or the drug candidate is immobilized on a solid phase,e.g., on a microtiter plate, by covalent or non-covalent attachments.Non-covalent attachment generally is accomplished by coating the solidsurface with a solution of the PRO226, PRO257, PRO268, PRO290, PRO36006,PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125, PRO1134,PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433,PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341,PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737,PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873,PRO10196, PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 orPRO85161 polypeptide and drying. Alternatively, an immobilized antibody,e.g., a monoclonal antibody, specific for the PRO226, PRO257, PRO268,PRO290, PRO36006, PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071,PRO1125, PRO1134, PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387,PRO1419, PRO1433, PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904,PRO35193, PRO4341, PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985,PRO4989, PRO5737, PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821,PRO9852, PRO9873, PRO10196, PRO34778, PRO20233, PRO21956, PRO57290,PRO38465, PRO38683 or PRO85161 polypeptide to be immobilized can be usedto anchor it to a solid surface. The assay is performed by adding thenon-immobilized component, which may be labeled by a detectable label,to the immobilized component, e.g., the coated surface containing theanchored component. When the reaction is complete, the non-reactedcomponents are removed, e.g., by washing, and complexes anchored on thesolid surface are detected. When the originally non-immobilizedcomponent carries a detectable label, the detection of label immobilizedon the surface indicates that complexing occurred. Where the originallynon-immobilized component does not carry a label, complexing can bedetected, for example, by using a labeled antibody specifically bindingthe immobilized complex.

If the candidate compound interacts with but does not bind to aparticular PRO226, PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365,PRO382, PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281,PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550,PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369,PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993,PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196,PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161polypeptide encoded by a gene identified herein, its interaction withthat polypeptide can be assayed by methods well known for detectingprotein-protein interactions. Such assays include traditionalapproaches, such as, e.g., cross-linking, co-immunoprecipitation, andco-purification through gradients or chromatographic columns. Inaddition, protein-protein interactions can be monitored by using ayeast-based genetic system described by Fields and co-workers (Fieldsand Song, Nature (London), 340:245-246 (1989); Chien et al., Proc. Natl.Acad. Sci. USA, 88:9578-9582 (1991)) as disclosed by Chevray andNathans, Proc. Natl. Acad. Sci. USA, 89: 5789-5793 (1991). Manytranscriptional activators, such as yeast GAL4, consist of twophysically discrete modular domains, one acting as the DNA-bindingdomain, the other one functioning as the transcription-activationdomain. The yeast expression system described in the foregoingpublications (generally referred to as the “two-hybrid system”) takesadvantage of this property, and employs two hybrid proteins, one inwhich the target protein is fused to the DNA-binding domain of GAL4, andanother, in which candidate activating proteins are fused to theactivation domain. The expression of a GAL1-lacZ reporter gene undercontrol of a GAL4-activated promoter depends on reconstitution of GAL4activity via protein-protein interaction. Colonies containinginteracting polypeptides are detected with a chromogenic substrate forβ-galactosidase. A complete kit (MATCHMAKER™) for identifyingprotein-protein interactions between two specific proteins using thetwo-hybrid technique is commercially available from Clontech. Thissystem can also be extended to map protein domains involved in specificprotein interactions as well as to pinpoint amino acid residues that arecrucial for these interactions.

Compounds that interfere with the interaction of a gene encoding aPRO226, PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382,PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343,PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571,PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381,PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017,PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196, PRO34778,PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161 polypeptideidentified herein and other intra- or extracellular components can betested as follows: usually a reaction mixture is prepared containing theproduct of the gene and the intra- or extracellular component underconditions and for a time allowing for the interaction and binding ofthe two products. To test the ability of a candidate compound to inhibitbinding, the reaction is run in the absence and in the presence of thetest compound. In addition, a placebo may be added to a third reactionmixture, to serve as positive control. The binding (complex formation)between the test compound and the intra- or extracellular componentpresent in the mixture is monitored as described hereinabove. Theformation of a complex in the control reaction(s) but not in thereaction mixture containing the test compound indicates that the testcompound interferes with the interaction of the test compound and itsreaction partner.

To assay for antagonists, the PRO226, PRO257, PRO268, PRO290, PRO36006,PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125, PRO1134,PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433,PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341,PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737,PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873,PRO10196, PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 orPRO85161 polypeptide may be added to a cell along with the compound tobe screened for a particular activity and the ability of the compound toinhibit the activity of interest in the presence of the PRO226, PRO257,PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382, PRO444, PRO705,PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343, PRO1379, PRO1380,PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571, PRO1572, PRO1759,PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381, PRO4407, PRO4425,PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017, PRO7174, PRO9744,PRO9821, PRO9852, PRO9873, PRO10196, PRO34778, PRO20233, PRO21956,PRO57290, PRO38465, PRO38683 or PRO85161 polypeptide indicates that thecompound is an antagonist to the PRO226, PRO257, PRO268, PRO290,PRO36006, PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125,PRO1134, PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419,PRO1433, PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193,PRO4341, PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989,PRO5737, PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852,PRO9873, PRO10196, PRO34778, PRO20233, PRO21956, PRO57290, PRO38465,PRO38683 or PRO85161 polypeptide. Alternatively, antagonists may bedetected by combining the PRO226, PRO257, PRO268, PRO290, PRO36006,PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125, PRO1134,PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433,PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341,PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737,PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873,PRO10196, PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 orPRO85161 polypeptide and a potential antagonist with membrane-boundPRO226, PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382,PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343,PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571,PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381,PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017,PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196, PRO34778,PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161 polypeptidereceptors or recombinant receptors under appropriate conditions for acompetitive inhibition assay. The PRO226, PRO257, PRO268, PRO290,PRO36006, PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125,PRO1134, PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419,PRO1433, PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193,PRO4341, PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989,PRO5737, PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852,PRO9873, PRO10196, PRO34778, PRO20233, PRO21956, PRO57290, PRO38465,PRO38683 or PRO85161 polypeptide can be labeled, such as byradioactivity, such that the number of PRO226, PRO257, PRO268, PRO290,PRO36006, PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125,PRO1134, PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419,PRO1433, PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193,PRO4341, PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989,PRO5737, PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852,PRO9873, PRO10196, PRO34778, PRO20233, PRO21956, PRO57290, PRO38465,PRO38683 or PRO85161 polypeptide molecules bound to the receptor can beused to determine the effectiveness of the potential antagonist. Thegene encoding the receptor can be identified by numerous methods knownto those of skill in the art, for example, ligand panning and FACSsorting. Coligan et al., Current Protocols in Immun., 1(2): Chapter 5(1991). Preferably, expression cloning is employed whereinpolyadenylated RNA is prepared from a cell responsive to the PRO226,PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382, PRO444,PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343, PRO1379,PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571, PRO1572,PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381, PRO4407,PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017, PRO7174,PRO9744, PRO9821, PRO9852, PRO9873, PRO10196, PRO34778, PRO20233,PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161 polypeptide and acDNA library created from this RNA is divided into pools and used totransfect COS cells or other cells that are not responsive to thePRO226, PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382,PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343,PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571,PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381,PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017,PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196, PRO34778,PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161polypeptide. Transfected cells that are grown on glass slides areexposed to labeled PRO226, PRO257, PRO268, PRO290, PRO36006, PRO363,PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155,PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474,PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348,PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800,PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196,PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161polypeptide. The PRO226, PRO257, PRO268, PRO290, PRO36006, PRO363,PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155,PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474,PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348,PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800,PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196,PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161polypeptide can be labeled by a variety of means including iodination orinclusion of a recognition site for a site-specific protein kinase.Following fixation and incubation, the slides are subjected toautoradiographic analysis. Positive pools are identified and sub-poolsare prepared and re-transfected using an interactive sub-pooling andre-screening process, eventually yielding a single clone that encodesthe putative receptor.

As an alternative approach for receptor identification, the labeledPRO226, PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382,PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343,PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571,PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381,PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017,PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196, PRO34778,PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161 polypeptidecan be photoaffinity-linked with cell membrane or extract preparationsthat express the receptor molecule. Cross-linked material is resolved byPAGE and exposed to X-ray film. The labeled complex containing thereceptor can be excised, resolved into peptide fragments, and subjectedto protein micro-sequencing. The amino acid sequence obtained frommicro-sequencing would be used to design a set of degenerateoligonucleotide probes to screen a cDNA library to identify the geneencoding the putative receptor.

Another approach in assessing the effect of an antagonist to a PRO226,PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382, PRO444,PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343, PRO1379,PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571, PRO1572,PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381, PRO4407,PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017, PRO7174,PRO9744, PRO9821, PRO9852, PRO9873, PRO10196, PRO34778, PRO20233,PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161 polypeptide, would beadministering a PRO226, PRO257, PRO268, PRO290, PRO36006, PRO363,PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155,PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474,PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348,PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800,PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196,PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161antagonist to a wild-type mouse in order to mimic a known knockoutphenotype. Thus, one would initially knockout the PRO226, PRO257,PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382, PRO444, PRO705,PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343, PRO1379, PRO1380,PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571, PRO1572, PRO1759,PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381, PRO4407, PRO4425,PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017, PRO7174, PRO9744,PRO9821, PRO9852, PRO9873, PRO10196, PRO34778, PRO20233, PRO21956,PRO57290, PRO38465, PRO38683 or PRO85161 gene of interest and observethe resultant phenotype as a consequence of knocking out or disruptingthe PRO226, PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382,PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343,PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571,PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381,PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017,PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196, PRO34778,PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161 gene.Subsequently, one could then assess the effectiveness of an antagonistto the PRO226, PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382,PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343,PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571,PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381,PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017,PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196, PRO34778,PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161 polypeptideby administering an antagonist to the PRO226, PRO257, PRO268, PRO290,PRO36006, PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125,PRO1134, PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419,PRO1433, PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193,PRO4341, PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989,PRO5737, PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852,PRO9873, PRO10196, PRO34778, PRO20233, PRO21956, PRO57290, PRO38465,PRO38683 or PRO85161 polypeptide to a wild-type mouse. An effectiveantagonist would be expected to mimic the phenotypic effect that wasinitially observed in the knockout animal.

Likewise, one could assess the effect of an agonist to a PRO226, PRO257,PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382, PRO444, PRO705,PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343, PRO1379, PRO1380,PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571, PRO1572, PRO1759,PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381, PRO4407, PRO4425,PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017, PRO7174, PRO9744,PRO9821, PRO9852, PRO9873, PRO10196, PRO34778, PRO20233, PRO21956,PRO57290, PRO38465, PRO38683 or PRO85161 polypeptide, by administering aPRO226, PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382,PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343,PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571,PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381,PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017,PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196, PRO34778,PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161 agonist toa non-human transgenic mouse in order to ameliorate a known negativeknockout phenotype. Thus, one would initially knockout the PRO226,PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382, PRO444,PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343, PRO1379,PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571, PRO1572,PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381, PRO4407,PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017, PRO7174,PRO9744, PRO9821, PRO9852, PRO9873, PRO10196, PRO34778, PRO20233,PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161 gene of interest andobserve the resultant phenotype as a consequence of knocking out ordisrupting the PRO226, PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365,PRO382, PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281,PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550,PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369,PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993,PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196,PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161gene. Subsequently, one could then assess the effectiveness of anagonist to the PRO226, PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365,PRO382, PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281,PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550,PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369,PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993,PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196,PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161polypeptide by administering an agonist to the PRO226, PRO257, PRO268,PRO290, PRO36006, PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071,PRO1125, PRO1134, PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387,PRO1419, PRO1433, PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904,PRO35193, PRO4341, PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985,PRO4989, PRO5737, PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821,PRO9852, PRO9873, PRO10196, PRO34778, PRO20233, PRO21956, PRO57290,PRO38465, PRO38683 or PRO85161 polypeptide to a the non-human transgenicmouse. An effective agonist would be expected to ameliorate the negativephenotypic effect that was initially observed in the knockout animal.

In another assay for antagonists, mammalian cells or a membranepreparation expressing the receptor would be incubated with a labeledPRO226, PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382,PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343,PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571,PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381,PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017,PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196, PRO34778,PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161 polypeptidein the presence of the candidate compound. The ability of the compoundto enhance or block this interaction could then be measured.

More specific examples of potential antagonists include anoligonucleotide that binds to the fusions of immunoglobulin with thePRO226, PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382,PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343,PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571,PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381,PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017,PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196, PRO34778,PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161polypeptide, and, in particular, antibodies including, withoutlimitation, poly- and monoclonal antibodies and antibody fragments,single-chain antibodies, anti-idiotypic antibodies, and chimeric orhumanized versions of such antibodies or fragments, as well as humanantibodies and antibody fragments. Alternatively, a potential antagonistmay be a closely related protein, for example, a mutated form of thePRO226, PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382,PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343,PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571,PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381,PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017,PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196, PRO34778,PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161 polypeptidethat recognizes the receptor but imparts no effect, therebycompetitively inhibiting the action of the PRO226, PRO257, PRO268,PRO290, PRO36006, PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071,PRO1125, PRO1134, PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387,PRO1419, PRO1433, PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904,PRO35193, PRO4341, PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985,PRO4989, PRO5737, PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821,PRO9852, PRO9873, PRO10196, PRO34778, PRO20233, PRO21956, PRO57290,PRO38465, PRO38683 or PRO85161 polypeptide.

Another potential PRO226, PRO257, PRO268, PRO290, PRO36006, PRO363,PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155,PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474,PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348,PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800,PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196,PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161polypeptide antagonist is an antisense RNA or DNA construct preparedusing antisense technology, where, e.g., an antisense RNA or DNAmolecule acts to block directly the translation of mRNA by hybridizingto targeted mRNA and preventing protein translation. Antisensetechnology can be used to control gene expression through triple-helixformation or antisense DNA or RNA, both of which methods are based onbinding of a polynucleotide to DNA or RNA. For example, the 5′ codingportion of the polynucleotide sequence, which encodes the mature PRO226,PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382, PRO444,PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343, PRO1379,PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571, PRO1572,PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381, PRO4407,PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017, PRO7174,PRO9744, PRO9821, PRO9852, PRO9873, PRO10196, PRO34778, PRO20233,PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161 polypeptides herein,is used to design an antisense RNA oligonucleotide of from about 10 to40 base pairs in length. A DNA oligonucleotide is designed to becomplementary to a region of the gene involved in transcription (triplehelix—see Lee et al., Nucl. Acids Res., 6:3073 (1979); Cooney et al.,Science, 241: 456 (1988); Dervan et al., Science, 251:1360 (1991)),thereby preventing transcription and the production of the PRO226,PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382, PRO444,PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343, PRO1379,PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571, PRO1572,PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381, PRO4407,PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017, PRO7174,PRO9744, PRO9821, PRO9852, PRO9873, PRO10196, PRO34778, PRO20233,PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161 polypeptide. Theantisense RNA oligonucleotide hybridizes to the mRNA in vivo and blockstranslation of the mRNA molecule into the PRO226, PRO257, PRO268,PRO290, PRO36006, PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071,PRO1125, PRO1134, PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387,PRO1419, PRO1433, PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904,PRO35193, PRO4341, PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985,PRO4989, PRO5737, PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821,PRO9852, PRO9873, PRO10196, PRO34778, PRO20233, PRO21956, PRO57290,PRO38465, PRO38683 or PRO85161 polypeptide (antisense—Okano, Neurochem.,56:560 (1991); Oligodeoxynucleotides as Antisense Inhibitors of GeneExpression (CRC Press: Boca Raton, Fla., 1988). The oligonucleotidesdescribed above can also be delivered to cells such that the antisenseRNA or DNA may be expressed in vivo to inhibit production of the PRO226,PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382, PRO444,PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343, PRO1379,PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571, PRO1572,PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381, PRO4407,PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017, PRO7174,PRO9744, PRO9821, PRO9852, PRO9873, PRO10196, PRO34778, PRO20233,PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161 polypeptide. Whenantisense DNA is used, oligodeoxyribonucleotides derived from thetranslation-initiation site, e.g., between about −10 and +10 positionsof the target gene nucleotide sequence, are preferred.

Potential antagonists include small molecules that bind to the activesite, the receptor binding site, or growth factor or other relevantbinding site of the PRO226, PRO257, PRO268, PRO290, PRO36006, PRO363,PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155,PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474,PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348,PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800,PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196,PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161polypeptide, thereby blocking the normal biological activity of thePRO226, PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382,PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343,PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571,PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381,PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017,PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196, PRO34778,PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161polypeptide. Examples of small molecules include, but are not limitedto, small peptides or peptide-like molecules, preferably solublepeptides, and synthetic non-peptidyl organic or inorganic compounds.

Ribozymes are enzymatic RNA molecules capable of catalyzing the specificcleavage of RNA. Ribozymes act by sequence-specific hybridization to thecomplementary target RNA, followed by endonucleolytic cleavage. Specificribozyme cleavage sites within a potential RNA target can be identifiedby known techniques. For further details see, e.g., Rossi, CurrentBiology, 4:469-471 (1994), and PCT publication No. WO 97/33551(published Sep. 18, 1997).

Nucleic acid molecules in triple-helix formation used to inhibittranscription should be single-stranded and composed ofdeoxynucleotides. The base composition of these oligonucleotides isdesigned such that it promotes triple-helix formation via Hoogsteenbase-pairing rules, which generally require sizeable stretches ofpurines or pyrimidines on one strand of a duplex. For further detailssee, e.g., PCT publication No. WO 97/33551, supra.

These small molecules can be identified by anyone or more of thescreening assays discussed hereinabove and/or by any other screeningtechniques well known for those skilled in the art.

Diagnostic and therapeutic uses of the herein disclosed molecules mayalso be based upon the positive functional assay hits disclosed anddescribed below.

F. Anti-PRO226, Anti-PRO257, Anti-PRO268, Anti-PRO290, Anti-PRO36006,Anti-PRO363, Anti-PRO365, Anti-PRO382, Anti-PRO444, Anti-PRO705,Anti-PRO1071, Anti-PRO1125, Anti-PRO1134, Anti-PRO1155, Anti-PRO1281,Anti-PRO1343, Anti-PRO1379, Anti-PRO1380, Anti-PRO1387, Anti-PRO1419,Anti-PRO1433, Anti-PRO1474, Anti-PRO1550, Anti-PRO1571, Anti-PRO1572,Anti-PRO1759, Anti-PRO1904, Anti-PRO35193, Anti-PRO4341, Anti-PRO4348,Anti-PRO4369, Anti-PRO4381, Anti-PRO4407, Anti-PRO4425, Anti-PRO4985,Anti-PRO4989, Anti-PRO5737, Anti-PRO5800, Anti-PRO5993, Anti-PRO6017,Anti-PRO7174, Anti-PRO9744, Anti-PRO9821, Anti-PRO9852, Anti-PRO9873,Anti-PRO10196, Anti-PRO34778, Anti-PRO20233, Anti-PRO21956,Anti-PRO57290, Anti-PRO38465, Anti-PRO38683 or Anti-PRO85161, Antibodies

The present invention provides anti-PRO226, anti-PRO257, anti-PRO268,anti-PRO290, anti-PRO36006, anti-PRO363, anti-PRO365, anti-PRO382,anti-PRO444, anti-PRO705, anti-PRO1071, anti-PRO1125, anti-PRO1134,anti-PRO1155, anti-PRO1281, anti-PRO1343, anti-PRO1379, anti-PRO1380,anti-PRO1387, anti-PRO1419, anti-PRO1433, anti-PRO1474, anti-PRO1550,anti-PRO1571, anti-PRO1572, anti-PRO1759, anti-PRO1904, anti-PRO35193,anti-PRO4341, anti-PRO4348, anti-PRO4369, anti-PRO4381, anti-PRO4407,anti-PRO4425, anti-PRO4985, anti-PRO4989, anti-PRO5737, anti-PRO5800,anti-PRO5993, anti-PRO6017, anti-PRO7174, anti-PRO9744, anti-PRO9821,anti-PRO9852, anti-PRO9873, anti-PRO10196, anti-PRO34778, anti-5,PRO20233, anti-PRO21956, anti-PRO57290, anti-PRO38465, anti-PRO38683 oranti-PRO85161 antibodies which may find use herein as therapeutic and/ordiagnostic agents. Exemplary antibodies include polyclonal, monoclonal,humanized, bispecific, and heteroconjugate antibodies.

1. Polyclonal Antibodies

Polyclonal antibodies are preferably raised in animals by multiplesubcutaneous (sc) or intraperitoneal (ip) injections of the relevantantigen and an adjuvant. It may be useful to conjugate the relevantantigen (especially when synthetic peptides are used) to a protein thatis immunogenic in the species to be immunized. For example, the antigencan be conjugated to keyhole limpet hemocyanin (KLH), serum albumin,bovine thyroglobulin, or soybean trypsin inhibitor, using a bifunctionalor derivatizing agent, e.g., maleimidobenzoyl sulfosuccinimide ester(conjugation through cysteine residues), N-hydroxysuccinimide (throughlysine residues), glutaraldehyde, succinic anhydride, SOCl₂, orR¹N═C═NR, where R and R¹ are different alkyl groups.

Animals are immunized against the antigen, immunogenic conjugates, orderivatives by combining, e.g., 100 μg or 5 μg of the protein orconjugate (for rabbits or mice, respectively) with 3 volumes of Freund'scomplete adjuvant and injecting the solution intradermally at multiplesites. One month later, the animals are boosted with ⅕ to 1/10 theoriginal amount of peptide or conjugate in Freund's complete adjuvant bysubcutaneous injection at multiple sites. Seven to 14 days later, theanimals are bled and the serum is assayed for antibody titer. Animalsare boosted until the titer plateaus. Conjugates also can be made inrecombinant cell culture as protein fusions. Also, aggregating agentssuch as alum are suitably used to enhance the immune response.

2. Monoclonal Antibodies

Monoclonal antibodies may be made using the hybridoma method firstdescribed by Kohler et al., Nature, 256:495 (1975), or may be made byrecombinant DNA methods (U.S. Pat. No. 4,816,567).

In the hybridoma method, a mouse or other appropriate host animal, suchas a hamster, is immunized as described above to elicit lymphocytes thatproduce or are capable of producing antibodies that will specificallybind to the protein used for immunization. Alternatively, lymphocytesmay be immunized in vitro. After immunization, lymphocytes are isolatedand then fused with a myeloma cell line using a suitable fusing agent,such as polyethylene glycol, to form a hybridoma cell (Goding,Monoclonal Antibodies: Principles and Practice, pp. 59-103 (AcademicPress, 1986)).

The hybridoma cells thus prepared are seeded and grown in a suitableculture medium which medium preferably contains one or more substancesthat inhibit the growth or survival of the unfused, parental myelomacells (also referred to as fusion partner). For example, if the parentalmyeloma cells lack the enzyme hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT), the selective culture medium for thehybridomas typically will include hypoxanthine, aminopterin, andthymidine (HAT medium), which substances prevent the growth ofHGPRT-deficient cells.

Preferred fusion partner myeloma cells are those that fuse efficiently,support stable high-level production of antibody by the selectedantibody-producing cells, and are sensitive to a selective medium thatselects against the unfused parental cells. Preferred myeloma cell linesare murine myeloma lines, such as those derived from MOPC-21 and MPC-11mouse tumors available from the Salk Institute Cell Distribution Center,San Diego, Calif. USA, and SP-2 and derivatives e.g., X63-Ag8-653 cellsavailable from the American Type Culture Collection, Manassas, Va., USA.Human myeloma and mouse-human heteromyeloma cell lines also have beendescribed for the production of human monoclonal antibodies (Kozbor, J.Immunol., 133:3001 (1984); and Brodeur et al., Monoclonal AntibodyProduction Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc.,New York, 1987)).

Culture medium in which hybridoma cells are growing is assayed forproduction of monoclonal antibodies directed against the antigen.Preferably, the binding specificity of monoclonal antibodies produced byhybridoma cells is determined by immunoprecipitation or by an in vitrobinding assay, such as radioimmunoassay (RIA) or enzyme-linkedimmunosorbent assay (ELISA).

The binding affinity of the monoclonal antibody can, for example, bedetermined by the Scatchard analysis described in Munson et al., Anal.Biochem., 107:220 (1980).

Once hybridoma cells that produce antibodies of the desired specificity,affinity, and/or activity are identified, the clones may be subcloned bylimiting dilution procedures and grown by standard methods (Goding,Monoclonal Antibodies: Principles and Practice, pp. 59-103 (AcademicPress, 1986)). Suitable culture media for this purpose include, forexample, D-MEM or RPMI-1640 medium. In addition, the hybridoma cells maybe grown in vivo as ascites tumors in an animal e.g., by i.p. injectionof the cells into mice.

The monoclonal antibodies secreted by the subclones are suitablyseparated from the culture medium, ascites fluid, or serum byconventional antibody purification procedures such as, for example,affinity chromatography (e.g., using protein A or protein G-Sepharose)or ion-exchange chromatography, hydroxylapatite chromatography, gelelectrophoresis, dialysis, etc.

DNA encoding the monoclonal antibodies is readily isolated and sequencedusing conventional procedures (e.g., by using oligonucleotide probesthat are capable of binding specifically to genes encoding the heavy andlight chains of murine antibodies). The hybridoma cells serve as apreferred source of such DNA. Once isolated, the DNA may be placed intoexpression vectors, which are then transfected into host cells such asE. coli cells, simian COS cells, Chinese Hamster Ovary (CHO) cells, ormyeloma cells that do not otherwise produce antibody protein, to obtainthe synthesis of monoclonal antibodies in the recombinant host cells.Review articles on recombinant expression in bacteria of DNA encodingthe antibody include Skerra et al., Curr. Opinion in Immunol., 5:256-262(1993) and Plückthun, Immunol. Revs. 130:151-188 (1992).

Monoclonal antibodies or antibody fragments can be isolated fromantibody phage libraries generated using the techniques described inMcCafferty et al., Nature, 348:552-554 (1990). Clackson et al., Nature,352:624-628 (1991) and Marks et al., J. Mol. Biol., 222:581-597 (1991)describe the isolation of murine and human antibodies, respectively,using phage libraries. Subsequent publications describe the productionof high affinity (nM range) human antibodies by chain shuffling (Markset al., Bio/Technology, 10:779-783 (1992)), as well as combinatorialinfection and in vivo recombination as a strategy for constructing verylarge phage libraries (Waterhouse et al., Nuc. Acids. Res. 21:2265-2266(1993)). Thus, these techniques are viable alternatives to traditionalmonoclonal antibody hybridoma techniques for isolation of monoclonalantibodies.

The DNA that encodes the antibody may be modified to produce chimeric orfusion antibody polypeptides, for example, by substituting human heavychain and light chain constant domain (C_(H) and C_(L)) sequences forthe homologous murine sequences (U.S. Pat. No. 4,816,567; and Morrison,et al., Proc. Natl Acad. Sci. USA, 81:6851 (1984)), or by fusing theimmunoglobulin coding sequence with all or part of the coding sequencefor a non-immunoglobulin polypeptide (heterologous polypeptide). Thenon-immunoglobulin polypeptide sequences can substitute for the constantdomains of an antibody, or they are substituted for the variable domainsof one antigen-combining site of an antibody to create a chimericbivalent antibody comprising one antigen-combining site havingspecificity for an antigen and another antigen-combining site havingspecificity for a different antigen.

3. Human and Humanized Antibodies

The anti-PRO226, anti-PRO257, anti-PRO268, anti-PRO290, anti-PRO36006,anti-PRO363, anti-PRO365, anti-PRO382, anti-PRO444, anti-PRO705,anti-PRO1071, anti-PRO1125, anti-PRO1134, anti-PRO1155, anti-PRO1281,anti-PRO1343, anti-PRO1379, anti-PRO1380, anti-PRO1387, anti-PRO1419,anti-PRO1433, anti-PRO1474, anti-PRO1550, anti-PRO1571, anti-PRO1572,anti-PRO1759, anti-PRO1904, anti-PRO35193, anti-PRO4341, anti-PRO4348,anti-PRO4369, anti-PRO4381, anti-PRO4407, anti-PRO4425, anti-PRO4985,anti-PRO4989, anti-PRO5737, anti-PRO5800, anti-PRO5993, anti-PRO6017,anti-PRO7174, anti-PRO9744, anti-PRO9821, anti-PRO9852, anti-PRO9873,anti-PRO10196, anti-PRO34778, anti-PRO20233, anti-PRO21956,anti-PRO57290, anti-PRO38465, anti-PRO38683 or anti-PRO85161 antibodiesof the invention may further comprise humanized antibodies or humanantibodies. Humanized forms of non-human (e.g., murine) antibodies arechimeric immunoglobulins, immunoglobulin chains or fragments thereof(such as Fv, Fab, Fab′, F(ab′)₂ or other antigen-binding subsequences ofantibodies) which contain minimal sequence derived from non-humanimmunoglobulin. Humanized antibodies include human immunoglobulins(recipient antibody) in which residues from a complementary determiningregion (CDR) of the recipient are replaced by residues from a CDR of anon-human species (donor antibody) such as mouse, rat or rabbit havingthe desired specificity, affinity and capacity. In some instances, Fvframework residues of the human immunoglobulin are replaced bycorresponding non-human residues. Humanized antibodies may also compriseresidues which are found neither in the recipient antibody nor in theimported CDR or framework sequences. In general, the humanized antibodywill comprise substantially all of at least one, and typically two,variable domains, in which all or substantially all of the CDR regionscorrespond to those of a non-human immunoglobulin and all orsubstantially all of the FR regions are those of a human immunoglobulinconsensus sequence. The humanized antibody optimally also will compriseat least a portion of an immunoglobulin constant region (Fc), typicallythat of a human immunoglobulin [Jones et al., Nature, 321:522-525(1986); Riechmann et al., Nature, 332:323-329 (1988); and Presta, Curr.Op. Struct. Biol., 2:593-596 (1992)].

Methods for humanizing non-human antibodies are well known in the art.Generally, a humanized antibody has one or more amino acid residuesintroduced into it from a source which is non-human. These non-humanamino acid residues are often referred to as “import” residues, whichare typically taken from an “import” variable domain. Humanization canbe essentially performed following the method of Winter and co-workers[Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature,332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)], bysubstituting rodent CDRs or CDR sequences for the correspondingsequences of a human antibody. Accordingly, such “humanized” antibodiesare chimeric antibodies (U.S. Pat. No. 4,816,567), wherein substantiallyless than an intact human variable domain has been substituted by thecorresponding sequence from a non-human species. In practice, humanizedantibodies are typically human antibodies in which some CDR residues andpossibly some FR residues are substituted by residues from analogoussites in rodent antibodies.

The choice of human variable domains, both light and heavy, to be usedin making the humanized antibodies is very important to reduceantigenicity and HAMA response (human anti-mouse antibody) when theantibody is intended for human therapeutic use. According to theso-called “best-fit” method, the sequence of the variable domain of arodent antibody is screened against the entire library of known humanvariable domain sequences. The human V domain sequence which is closestto that of the rodent is identified and the human framework region (FR)within it accepted for the humanized antibody (Sims et al., J. Immunol.151:2296 (1993); Chothia et al., J. Mol. Biol., 196:901 (1987)). Anothermethod uses a particular framework region derived from the consensussequence of all human antibodies of a particular subgroup of light orheavy chains. The same framework may be used for several differenthumanized antibodies (Carter et al., Proc. Natl. Acad. Sci. USA, 89:4285(1992); Presta et al., J. Immunol. 151:2623 (1993)).

It is further important that antibodies be humanized with retention ofhigh binding affinity for the antigen and other favorable biologicalproperties. To achieve this goal, according to a preferred method,humanized antibodies are prepared by a process of analysis of theparental sequences and various conceptual humanized products usingthree-dimensional models of the parental and humanized sequences.Three-dimensional immunoglobulin models are commonly available and arefamiliar to those skilled in the art. Computer programs are availablewhich illustrate and display probable three-dimensional conformationalstructures of selected candidate immunoglobulin sequences. Inspection ofthese displays permits analysis of the likely role of the residues inthe functioning of the candidate immunoglobulin sequence, i.e., theanalysis of residues that influence the ability of the candidateimmunoglobulin to bind its antigen. In this way, FR residues can beselected and combined from the recipient and import sequences so thatthe desired antibody characteristic, such as increased affinity for thetarget antigen(s), is achieved. In general, the hypervariable regionresidues are directly and most substantially involved in influencingantigen binding.

Various forms of a humanized anti-PRO226, anti-PRO257, anti-PRO268,anti-PRO290, anti-PRO36006, anti-PRO363, anti-PRO365, anti-PRO382,anti-PRO444, anti-PRO705, anti-PRO1071, anti-PRO1125, anti-PRO1134,anti-PRO1155, anti-PRO1281, anti-PRO1343, anti-PRO1379, anti-PRO1380,anti-PRO1387, anti-PRO1419, anti-PRO1433, anti-PRO1474, anti-PRO1550,anti-PRO1571, anti-PRO1572, anti-PRO1759, anti-PRO1904, anti-PRO35193,anti-PRO4341, anti-PRO4348, anti-PRO4369, anti-PRO4381, anti-PRO4407,anti-PRO4425, anti-PRO4985, anti-PRO4989, anti-PRO5737, anti-PRO5800,anti-PRO5993, anti-PRO6017, anti-PRO7174, anti-PRO9744, anti-PRO9821,anti-PRO9852, anti-PRO9873, anti-PRO10196, anti-PRO34778, anti-PRO20233,anti-PRO21956, anti-PRO57290, anti-PRO38465, anti-PRO38683 oranti-PRO85161 antibody are contemplated. For example, the humanizedantibody may be an antibody fragment, such as a Fab, which is optionallyconjugated with one or more cytotoxic agent(s) in order to generate animmunoconjugate. Alternatively, the humanized antibody may be an intactantibody, such as an intact IgG1 antibody.

As an alternative to humanization, human antibodies can be generated.For example, it is now possible to produce transgenic animals (e.g.,mice) that are capable, upon immunization, of producing a fullrepertoire of human antibodies in the absence of endogenousimmunoglobulin production. For example, it has been described that thehomozygous deletion of the antibody heavy-chain joining region (J_(H))gene in chimeric and germ-line mutant mice results in completeinhibition of endogenous antibody production. Transfer of the humangerm-line immunoglobulin gene array into such germ-line mutant mice willresult in the production of human antibodies upon antigen challenge.See, e.g., Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90:2551(1993); Jakobovits et al., Nature, 362:255-258 (1993); Bruggemann etal., Year in Immuno. 7:33 (1993); U.S. Pat. Nos. 5,545,806, 5,569,825,5,591,669 (all of GenPharm); U.S. Pat. No. 5,545,807; and WO 97/17852.

Alternatively, phage display technology (McCafferty et al., Nature348:552-553 [1990]) can be used to produce human antibodies and antibodyfragments in vitro, from immunoglobulin variable (V) domain generepertoires from unimmunized donors. According to this technique,antibody V domain genes are cloned in-frame into either a major or minorcoat protein gene of a filamentous bacteriophage, such as M13 or fd, anddisplayed as functional antibody fragments on the surface of the phageparticle. Because the filamentous particle contains a single-strandedDNA copy of the phage genome, selections based on the functionalproperties of the antibody also result in selection of the gene encodingthe antibody exhibiting those properties. Thus, the phage mimics some ofthe properties of the B-cell. Phage display can be performed in avariety of formats, reviewed in, e.g., Johnson, Kevin S, and Chiswell,David J., Current Opinion in Structural Biology 3:564-571 (1993).Several sources of V-gene segments can be used for phage display.Clackson et al., Nature, 352:624-628 (1991) isolated a diverse array ofanti-oxazolone antibodies from a small random combinatorial library of Vgenes derived from the spleens of immunized mice. A repertoire of Vgenes from unimmunized human donors can be constructed and antibodies toa diverse array of antigens (including self-antigens) can be isolatedessentially following the techniques described by Marks et al., J. Mol.Biol. 222:581-597 (1991), or Griffith et al., EMBO J. 12:725-734 (1993).See, also, U.S. Pat. Nos. 5,565,332 and 5,573,905.

As discussed above, human antibodies may also be generated by in vitroactivated B cells (see U.S. Pat. Nos. 5,567,610 and 5,229,275).

4. Antibody Fragments

In certain circumstances there are advantages of using antibodyfragments, rather than whole antibodies. The smaller size of thefragments allows for rapid clearance, and may lead to improved access tosolid tumors.

Various techniques have been developed for the production of antibodyfragments. Traditionally, these fragments were derived via proteolyticdigestion of intact antibodies (see, e.g., Morimoto et al., Journal ofBiochemical and Biophysical Methods 24:107-117 (1992); and Brennan etal., Science, 229:81 (1985)). However, these fragments can now beproduced directly by recombinant host cells. Fab, Fv and ScFv antibodyfragments can all be expressed in and secreted from E. coli, thusallowing the facile production of large amounts of these fragments.Antibody fragments can be isolated from the antibody phage librariesdiscussed above. Alternatively, Fab′-SH fragments can be directlyrecovered from E. coli and chemically coupled to form F(ab′)₂ fragments(Carter et al., Bio/Technology 10:163-167 (1992)). According to anotherapproach, F(ab′)₂ fragments can be isolated directly from recombinanthost cell culture. Fab and F(ab′)₂ fragment with increased in vivohalf-life comprising a salvage receptor binding epitope residues aredescribed in U.S. Pat. No. 5,869,046. Other techniques for theproduction of antibody fragments will be apparent to the skilledpractitioner. The antibody of choice is a single chain Fv fragment(scFv). See WO 93/16185; U.S. Pat. No. 5,571,894; and U.S. Pat. No.5,587,458. Fv and sFv are the only species with intact combining sitesthat are devoid of constant regions; thus, they are suitable for reducednonspecific binding during in vivo use. sFv fusion proteins may beconstructed to yield fusion of an effector protein at either the aminoor the carboxy terminus of an sFv. See Antibody Engineering, ed.Borrebaeck, supra. The antibody fragment may also be a “linearantibody”, e.g., as described in U.S. Pat. No. 5,641,870 for example.Such linear antibody fragments may be monospecific or bispecific.

5. Bispecific Antibodies

Bispecific antibodies are antibodies that have binding specificities forat least two different epitopes. Exemplary bispecific antibodies maybind to two different epitopes of a PRO226, PRO257, PRO268, PRO290,PRO36006, PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125,PRO1134, PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419,PRO1433, PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193,PRO4341, PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989,PRO5737, PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852,PRO9873, PRO10196, PRO34778, PRO20233, PRO21956, PRO57290, PRO38465,PRO38683 or PRO85161 protein as described herein. Other such antibodiesmay combine a PRO226, PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365,PRO382, PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281,PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550,PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369,PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993,PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196,PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161binding site with a binding site for another protein. Alternatively, ananti-PRO226, anti-PRO257, anti-PRO268, anti-PRO290, anti-PRO36006,anti-PRO363, anti-PRO365, anti-PRO382, anti-PRO444, anti-PRO705,anti-PRO1071, anti-PRO1125, anti-PRO1134, anti-PRO1155, anti-PRO1281,anti-PRO1343, anti-PRO1379, anti-PRO1380, anti-PRO1387, anti-PRO1419,anti-PRO1433, anti-PRO1474, anti-PRO1550, anti-PRO1571, anti-PRO1572,anti-PRO1759, anti-PRO1904, anti-PRO35193, anti-PRO4341, anti-PRO4348,anti-PRO4369, anti-PRO4381, anti-PRO4407, anti-PRO4425, anti-PRO4985,anti-PRO4989, anti-PRO5737, anti-PRO5800, anti-PRO5993, anti-PRO6017,anti-PRO7174, anti-PRO9744, anti-PRO9821, anti-PRO9852, anti-PRO9873,anti-PRO10196, anti-PRO34778, anti-PRO20233, anti-PRO21956,anti-PRO57290, anti-PRO38465, anti-PRO38683 or anti-PRO85161 arm may becombined with an arm which binds to a triggering molecule on a leukocytesuch as a T-cell receptor molecule (e.g. CD3), or Fc receptors for IgG(FcγR), such as FcγRI (CD64), FcγRII (CD32) and FcγRIII (CD16), so as tofocus and localize cellular defense mechanisms to the PRO226-, PRO257-,PRO268-, PRO290-, PRO36006-, PRO363-, PRO365-, PRO382-, PRO444-,PRO705-, PRO1071-, PRO1125-, PRO1134-, PRO1155-, PRO1281-, PRO1343-,PRO1379-, PRO1380-, PRO1387-, PRO1419-, PRO1433-, PRO1474-, PRO1550-,PRO1571-, PRO1572-, PRO1759-, PRO1904-, PRO35193-, PRO4341-, PRO4348-,PRO4369-, PRO4381-, PRO4407-, PRO4425-, PRO4985-, PRO4989-, PRO5737-,PRO5800-, PRO5993-, PRO6017-, PRO7174-, PRO9744-, PRO9821-, PRO9852-,PRO9873-, PRO10196-, PRO34778-, PRO20233-, PRO21956-, PRO57290-,PRO38465-, PRO38683- or PRO85161-expressing cell. Bispecific antibodiesmay also be used to localize cytotoxic agents to cells which express aPRO226, PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382,PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343,PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571,PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381,PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017,PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196, PRO34778,PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161polypeptide. These antibodies possess a PRO226-, PRO257-, PRO268-,PRO290-, PRO36006-, PRO363-, PRO365-, PRO382-PRO444-, PRO705-, PRO1071-,PRO1125-, PRO1134-, PRO1155-, PRO1281-, PRO1343-, PRO1379-, PRO1380-,PRO1387-, PRO1419-, PRO1433-, PRO1474-, PRO1550-, PRO1571-, PRO1572-,PRO1759-, PRO1904-, PRO35193-, PRO4341-, PRO4348-, PRO4369-, PRO4381-,PRO4407-, PRO4425-, PRO4985-, PRO4989-, PRO5737-, PRO5800-, PRO5993-,PRO6017-, PRO7174-, PRO9744-, PRO9821-, PRO9852-, PRO9873-, PRO10196-,PRO34778-, PRO20233-, PRO21956-, PRO57290-, PRO38465-, PRO38683- orPRO85161-binding arm and an arm which binds the cytotoxic agent (e.g.,saporin, anti-interferon-α, vinca alkaloid, ricin A chain, methotrexateor radioactive isotope hapten). Bispecific antibodies can be prepared asfull length antibodies or antibody fragments (e.g., F(ab′)₂ bispecificantibodies).

WO 96/16673 describes a bispecific anti-ErbB2/anti-FcγRIII antibody andU.S. Pat. No. 5,837,234 discloses a bispecific anti-ErbB2/anti-FcγRIantibody. A bispecific anti-ErbB2/Fcα antibody is shown in WO98/02463.U.S. Pat. No. 5,821,337 teaches a bispecific anti-ErbB2/anti-CD3antibody.

Methods for making bispecific antibodies are known in the art.Traditional production of full length bispecific antibodies is based onthe co-expression of two immunoglobulin heavy chain-light chain pairs,where the two chains have different specificities (Millstein et al.,Nature 305:537-539 (1983)). Because of the random assortment ofimmunoglobulin heavy and light chains, these hybridomas (quadromas)produce a potential mixture of 10 different antibody molecules, of whichonly one has the correct bispecific structure. Purification of thecorrect molecule, which is usually done by affinity chromatographysteps, is rather cumbersome, and the product yields are low. Similarprocedures are disclosed in WO 93/08829, and in Traunecker et al., EMBOJ. 10:3655-3659 (1991).

According to a different approach, antibody variable domains with thedesired binding specificity (antibody-antigen combining sites) are fusedto immunoglobulin constant domain sequences. Preferably, the fusion iswith an Ig heavy chain constant domain, comprising at least part of thehinge, C_(H)2, and C_(H)3 regions. It is preferred to have the firstheavy-chain constant region (C_(H)1) containing the site necessary forlight chain bonding, present in at least one of the fusions. DNAsencoding the immunoglobulin heavy chain fusions and, if desired, theimmunoglobulin light chain, are inserted into separate expressionvectors, and are co-transfected into a suitable host cell. This providesfor greater flexibility in adjusting the mutual proportions of the threepolypeptide fragments when unequal ratios of the three polypeptidechains used in the construction provide the optimum yield of the desiredbispecific antibody. It is, however, possible to insert the codingsequences for two or all three polypeptide chains into a singleexpression vector when the expression of at least two polypeptide chainsin equal ratios results in high yields or when the ratios have nosignificant affect on the yield of the desired chain combination.

The invention provides bispecific antibodies which are composed of ahybrid immunoglobulin heavy chain with a first binding specificity inone arm, and a hybrid immunoglobulin heavy chain-light chain pair(providing a second binding specificity) in the other arm. It was foundthat this asymmetric structure facilitates the separation of the desiredbispecific compound from unwanted immunoglobulin chain combinations, asthe presence of an immunoglobulin light chain in only one half of thebispecific molecule provides for a facile way of separation. Thisapproach is disclosed in WO 94/04690. For further details of generatingbispecific antibodies see, for example, Suresh et al., Methods inEnzymology 121:210 (1986).

According to another approach described in U.S. Pat. No. 5,731,168, theinterface between a pair of antibody molecules can be engineered tomaximize the percentage of heterodimers which are recovered fromrecombinant cell culture. The preferred interface comprises at least apart of the C_(H)3 domain. In this method, one or more small amino acidside chains from the interface of the first antibody molecule arereplaced with larger side chains (e.g., tyrosine or tryptophan).Compensatory “cavities” of identical or similar size to the large sidechain(s) are created on the interface of the second antibody molecule byreplacing large amino acid side chains with smaller ones (e.g., alanineor threonine). This provides a mechanism for increasing the yield of theheterodimer over other unwanted end-products such as homodimers.

Bispecific antibodies include cross-linked or “heteroconjugate”antibodies. For example, one of the antibodies in the heteroconjugatecan be coupled to avidin, the other to biotin. Such antibodies have, forexample, been proposed to target immune system cells to unwanted cells(U.S. Pat. No. 4,676,980), and for treatment of HIV infection (WO91/00360, WO 92/200373, and EP 03089). Heteroconjugate antibodies may bemade using any convenient cross-linking methods. Suitable cross-linkingagents are well known in the art, and are disclosed in U.S. Pat. No.4,676,980, along with a number of cross-linking techniques.

Techniques for generating bispecific antibodies from antibody fragmentshave also been described in the literature. For example, bispecificantibodies can be prepared using chemical linkage. Brennan et al.,Science 229:81 (1985) describe a procedure wherein intact antibodies areproteolytically cleaved to generate F(ab′)₂ fragments. These fragmentsare reduced in the presence of the dithiol complexing agent, sodiumarsenite, to stabilize vicinal dithiols and prevent intermoleculardisulfide formation. The Fab′ fragments generated are then converted tothionitrobenzoate (TNB) derivatives. One of the Fab′-TNB derivatives isthen reconverted to the Fab′-thiol by reduction with mercaptoethylamineand is mixed with an equimolar amount of the other Fab′-TNB derivativeto form the bispecific antibody. The bispecific antibodies produced canbe used as agents for the selective immobilization of enzymes.

Recent progress has facilitated the direct recovery of Fab′-SH fragmentsfrom E. coli, which can be chemically coupled to form bispecificantibodies. Shalaby et al., J. Exp. Med. 175: 217-225 (1992) describethe production of a fully humanized bispecific antibody F(ab′)₂molecule. Each Fab′ fragment was separately secreted from E. coli andsubjected to directed chemical coupling in vitro to form the bispecificantibody. The bispecific antibody thus formed was able to bind to cellsoverexpressing the ErbB2 receptor and normal human T cells, as well astrigger the lytic activity of human cytotoxic lymphocytes against humanbreast tumor targets. Various techniques for making and isolatingbispecific antibody fragments directly from recombinant cell culturehave also been described. For example, bispecific antibodies have beenproduced using leucine zippers. Kostelny et al., J. Immunol.148(5):1547-1553 (1992). The leucine zipper peptides from the Fos andJun proteins were linked to the Fab′ portions of two differentantibodies by gene fusion. The antibody homodimers were reduced at thehinge region to form monomers and then re-oxidized to form the antibodyheterodimers. This method can also be utilized for the production ofantibody homodimers. The “diabody” technology described by Hollinger etal., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993) has provided analternative mechanism for making bispecific antibody fragments. Thefragments comprise a V_(H) connected to a V_(L) by a linker which is tooshort to allow pairing between the two domains on the same chain.Accordingly, the V_(H) and V_(L) domains of one fragment are forced topair with the complementary V_(L) and V_(H) domains of another fragment,thereby forming two antigen-binding sites. Another strategy for makingbispecific antibody fragments by the use of single-chain Fv (sFv) dimershas also been reported. See Gruber et al., J. Immunol., 152:5368 (1994).

Antibodies with more than two valencies are contemplated. For example,trispecific antibodies can be prepared. Tutt et al., J. Immunol. 147:60(1991).

6. Heteroconjugate Antibodies

Heteroconjugate antibodies are also within the scope of the presentinvention. Heteroconjugate antibodies are composed of two covalentlyjoined antibodies. Such antibodies have, for example, been proposed totarget immune system cells to unwanted cells [U.S. Pat. No. 4,676,980],and for treatment of HIV infection [WO 91/00360; WO 92/200373; EP03089]. It is contemplated that the antibodies may be prepared in vitrousing known methods in synthetic protein chemistry, including thoseinvolving crosslinking agents. For example, immunotoxins may beconstructed using a disulfide exchange reaction or by forming athioether bond. Examples of suitable reagents for this purpose includeiminothiolate and methyl-4-mercaptobutyrimidate and those disclosed, forexample, in U.S. Pat. No. 4,676,980.

7. Multivalent Antibodies

A multivalent antibody may be internalized (and/or catabolized) fasterthan a bivalent antibody by a cell expressing an antigen to which theantibodies bind. The antibodies of the present invention can bemultivalent antibodies (which are other than of the IgM class) withthree or more antigen binding sites (e.g. tetravalent antibodies), whichcan be readily produced by recombinant expression of nucleic acidencoding the polypeptide chains of the antibody. The multivalentantibody can comprise a dimerization domain and three or more antigenbinding sites. The preferred dimerization domain comprises (or consistsof) an Fc region or a hinge region. In this scenario, the antibody willcomprise an Fc region and three or more antigen binding sitesamino-terminal to the Fc region. The preferred multivalent antibodyherein comprises (or consists of) three to about eight, but preferablyfour, antigen binding sites. The multivalent antibody comprises at leastone polypeptide chain (and preferably two polypeptide chains), whereinthe polypeptide chain(s) comprise two or more variable domains. Forinstance, the polypeptide chain(s) may compriseVD1-(X1)_(n)-VD2-(X2)_(n)-Fc, wherein VD1 is a first variable domain,VD2 is a second variable domain, Fc is one polypeptide chain of an Fcregion, X1 and X2 represent an amino acid or polypeptide, and n is 0or 1. For instance, the polypeptide chain(s) may comprise:VH-CH1-flexible linker-VH-CH1-Fc region chain; or VH-CH1-VH-CH1-Fcregion chain. The multivalent antibody herein preferably furthercomprises at least two (and preferably four) light chain variable domainpolypeptides. The multivalent antibody herein may, for instance,comprise from about two to about eight light chain variable domainpolypeptides. The light chain variable domain polypeptides contemplatedhere comprise alight chain variable domain and, optionally, furthercomprise a CL domain.

8. Effector Function Engineering

It may be desirable to modify the antibody of the invention with respectto effector function, e.g., so as to enhance antigen-dependentcell-mediated cytotoxicity (ADCC) and/or complement dependentcytotoxicity (CDC) of the antibody. This may be achieved by introducingone or more amino acid substitutions in an Fc region of the antibody.Alternatively or additionally, cysteine residue(s) may be introduced inthe Fc region, thereby allowing interchain disulfide bond formation inthis region. The homodimeric antibody thus generated may have improvedinternalization capability and/or increased complement-mediated cellkilling and antibody-dependent cellular cytotoxicity (ADCC). See Caronet al., J. Exp Med. 176:1191-1195 (1992) and Shopes, B. J. Immunol.148:2918-2922 (1992). Homodimeric antibodies with enhanced anti-tumoractivity may also be prepared using heterobifunctional cross-linkers asdescribed in Wolff et al., Cancer Research 53:2560-2565 (1993).Alternatively, an antibody can be engineered which has dual Fc regionsand may thereby have enhanced complement lysis and ADCC capabilities.See Stevenson et al., Anti-Cancer Drug Design 3:219-230 (1989). Toincrease the serum half life of the antibody, one may incorporate asalvage receptor binding epitope into the antibody (especially anantibody fragment) as described in U.S. Pat. No. 5,739,277, for example.As used herein, the term “salvage receptor binding epitope” refers to anepitope of the Fc region of an IgG molecule (e.g., IgG₁, IgG₂, IgG₃, orIgG₄) that is responsible for increasing the in vivo serum half-life ofthe IgG molecule.

9. Immunoconjugates

The invention also pertains to immunoconjugates comprising an antibodyconjugated to a cytotoxic agent such as a chemotherapeutic agent, agrowth inhibitory agent, a toxin (e.g., an enzymatically active toxin ofbacterial, fungal, plant, or animal origin, or fragments thereof), or aradioactive isotope (i.e., a radioconjugate).

Chemotherapeutic agents useful in the generation of suchimmunoconjugates have been described above. Enzymatically active toxinsand fragments thereof that can be used include diphtheria A chain,nonbinding active fragments of diphtheria toxin, exotoxin A chain (fromPseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain,alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolacaamericana proteins (PAPI, PAPII, and PAP-S), momordica charantiainhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin,mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes. Avariety of radionuclides are available for the production ofradioconjugated antibodies. Examples include ²¹²Bi, ¹³¹I, ¹³¹In, ⁹⁰Y,and ¹⁸⁶Re. Conjugates of the antibody and cytotoxic agent are made usinga variety of bifunctional protein-coupling agents such asN-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane(IT), bifunctional derivatives of imidoesters (such as dimethyladipimidate HCL), active esters (such as disuccinimidyl suberate),aldehydes (such as glutareldehyde), bis-azido compounds (such asbis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (such asbis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin canbe prepared as described in Vitetta et al, Science, 238: 1098 (1987).Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent forconjugation of radionucleotide to the antibody. See WO94/11026.

Conjugates of an antibody and one or more small molecule toxins, such asa calicheamicin, maytansinoids, a trichothene, and CC1065, and thederivatives of these toxins that have toxin activity, are alsocontemplated herein.

Maytansine and Maytansinoids

The invention provides an anti-PRO226, anti-PRO257, anti-PRO268,anti-PRO290, anti-PRO36006, anti-PRO363, anti-PRO365, anti-PRO382,anti-PRO444, anti-PRO705, anti-PRO1071, anti-PRO1125, anti-PRO1134,anti-PRO1155, anti-PRO1281, anti-PRO1343, anti-PRO1379, anti-PRO1380,anti-PRO1387, anti-PRO1419, anti-PRO1433, anti-PRO1474, anti-PRO1550,anti-PRO1571, anti-PRO1572, anti-PRO1759, anti-PRO1904, anti-PRO35193,anti-PRO4341, anti-PRO4348, anti-PRO4369, anti-PRO4381, anti-PRO4407,anti-PRO4425, anti-PRO4985, anti-PRO4989, anti-PRO5737, anti-PRO5800,anti-PRO5993, anti-PRO6017, anti-PRO7174, anti-PRO9744, anti-PRO9821,anti-PRO9852, anti-PRO9873, anti-PRO10196, anti-PRO34778, anti-PRO20233,anti-PRO21956, anti-PRO57290, anti-PRO38465, anti-PRO38683 oranti-PRO85161 antibody (full length or fragments) which is conjugated toone or more maytansinoid molecules.

Maytansinoids are mitototic inhibitors which act by inhibiting tubulinpolymerization. Maytansine was first isolated from the east Africanshrub Maytenus serrata (U.S. Pat. No. 3,896,111). Subsequently, it wasdiscovered that certain microbes also produce maytansinoids, such asmaytansinol and C-3 maytansinol esters (U.S. Pat. No. 4,151,042).Synthetic maytansinol and derivatives and analogues thereof aredisclosed, for example, in U.S. Pat. Nos. 4,137,230; 4,248,870;4,256,746; 4,260,608; 4,265,814; 4,294,757; 4,307,016; 4,308,268;4,308,269; 4,309,428; 4,313,946; 4,315,929; 4,317,821; 4,322,348;4,331,598; 4,361,650; 4,364,866; 4,424,219; 4,450,254; 4,362,663; and4,371,533, the disclosures of which are hereby expressly incorporated byreference.

Maytansinoid-Antibody Conjugates

In an attempt to improve their therapeutic index, maytansine andmaytansinoids have been conjugated to antibodies specifically binding totumor cell antigens. Immunoconjugates containing maytansinoids and theirtherapeutic use are disclosed, for example, in U.S. Pat. Nos. 5,208,020,5,416,064 and European Patent EP 0 425 235 B1, the disclosures of whichare hereby expressly incorporated by reference. Liu et al., Proc. Natl.Acad. Sci. USA 93:8618-8623 (1996) described immunoconjugates comprisinga maytansinoid designated DM1 linked to the monoclonal antibody C242directed against human colorectal cancer. The conjugate was found to behighly cytotoxic towards cultured colon cancer cells, and showedantitumor activity in an in vivo tumor growth assay. Chari et al.,Cancer Research 52:127-131 (1992) describe immunoconjugates in which amaytansinoid was conjugated via a disulfide linker to the murineantibody A7 binding to an antigen on human colon cancer cell lines, orto another murine monoclonal antibody TA.1 that binds the HER-2/neuoncogene. The cytotoxicity of the TA.1-maytansonoid conjugate was testedin vitro on the human breast cancer cell line SK-BR-3, which expresses3×10⁵ HER-2 surface antigens per cell. The drug conjugate achieved adegree of cytotoxicity similar to the free maytansonid drug, which couldbe increased by increasing the number of maytansinoid molecules perantibody molecule. The A7-maytansinoid conjugate showed low systemiccytotoxicity in mice.

Anti-PRO226, Anti-PRO257, Anti-PRO268, Anti-PRO290, Anti-PRO36006,Anti-PRO363, Anti-PRO365, Anti-PRO382, Anti-PRO444, Anti-PRO705,Anti-PRO1071, Anti-PRO1125, Anti-PRO1134, Anti-PRO1155, Anti-PRO1281,Anti-PRO1343, Anti-PRO1379, Anti-PRO1380, Anti-PRO1387, Anti-PRO1419,Anti-PRO1433, Anti-PRO1474, Anti-PRO1550, Anti-PRO1571, Anti-PRO1572,Anti-PRO1759, Anti-PRO1904, Anti-PRO35193, Anti-PRO4341, Anti-PRO4348,Anti-PRO4369, Anti-PRO4381, Anti-PRO4407, Anti-PRO4425, Anti-PRO4985,Anti-PRO4989, Anti-PRO5737, Anti-PRO5800, Anti-PRO5993, Anti-PRO6017,Anti-PRO7174, Anti-PRO9744, Anti-PRO9821, Anti-PRO9852, Anti-PRO9873,Anti-PRO10196, Anti-PRO34778, Anti-PRO20233, Anti-PRO21956,Anti-PRO57290, Anti-PRO38465, Anti-PRO38683 or Anti-PRO85161Antibody-Maytansinoid Conjugates (Immunoconjugates)

Anti-PRO226, anti-PRO257, anti-PRO268, anti-PRO290, anti-PRO36006,anti-PRO363, anti-PRO365, anti-PRO382, anti-PRO444, anti-PRO705,anti-PRO1071, anti-PRO1125, anti-PRO1134, anti-PRO1155, anti-PRO1281,anti-PRO1343, anti-PRO1379, anti-PRO1380, anti-PRO1387, anti-PRO1419,anti-PRO1433, anti-PRO1474, anti-PRO1550, anti-PRO1571, anti-PRO1572,anti-PRO1759, anti-PRO1904, anti-PRO35193, anti-PRO4341, anti-PRO4348,anti-PRO4369, anti-PRO4381, anti-PRO4407, anti-PRO4425, anti-PRO4985,anti-PRO4989, anti-PRO5737, anti-PRO5800, anti-PRO5993, anti-PRO6017,anti-PRO7174, anti-PRO9744, anti-PRO9821, anti-PRO9852, anti-PRO9873,anti-PRO10196, anti-PRO34778, anti-PRO20233, anti-PRO21956,anti-PRO57290, anti-PRO38465, anti-PRO38683 or anti-PRO85161antibody-maytansinoid conjugates are prepared by chemically linking ananti-PRO226, anti-PRO257, anti-PRO268, anti-PRO290, anti-PRO36006,anti-PRO363, anti-PRO365, anti-PRO382, anti-PRO444, anti-PRO705,anti-PRO1071, anti-PRO1125, anti-PRO1134, anti-PRO1155, anti-PRO1281,anti-PRO1343, anti-PRO1379, anti-PRO1380, anti-PRO1387, anti-PRO1419,anti-PRO1433, anti-PRO1474, anti-PRO1550, anti-PRO1571, anti-PRO1572,anti-PRO1759, anti-PRO1904, anti-PRO35193, anti-PRO4341, anti-PRO4348,anti-PRO4369, anti-PRO4381, anti-PRO4407, anti-PRO4425, anti-PRO4985,anti-PRO4989, anti-PRO5737, anti-PRO5800, anti-PRO5993, anti-PRO6017,anti-PRO7174, anti-PRO9744, anti-PRO9821, anti-PRO9852, anti-PRO9873,anti-PRO10196, anti-PRO34778, anti-PRO20233, anti-PRO21956,anti-PRO57290, anti-PRO38465, anti-PRO38683 or anti-PRO85161 antibody toa maytansinoid molecule without significantly diminishing the biologicalactivity of either the antibody or the maytansinoid molecule. An averageof 3-4 maytansinoid molecules conjugated per antibody molecule has shownefficacy in enhancing cytotoxicity of target cells without negativelyaffecting the function or solubility of the antibody, although even onemolecule of toxin/antibody would be expected to enhance cytotoxicityover the use of naked antibody. Maytansinoids are well known in the artand can be synthesized by known techniques or isolated from naturalsources. Suitable maytansinoids are disclosed, for example, in U.S. Pat.No. 5,208,020 and in the other patents and nonpatent publicationsreferred to hereinabove. Preferred maytansinoids are maytansinol andmaytansinol analogues modified in the aromatic ring or at otherpositions of the maytansinol molecule, such as various maytansinolesters.

There are many linking groups known in the art for makingantibody-maytansinoid conjugates, including, for example, thosedisclosed in U.S. Pat. No. 5,208,020 or EP Patent 0 425 235 B1, andChari et al., Cancer Research 52:127-131 (1992). The linking groupsinclude disulfide groups, thioether groups, acid labile groups,photolabile groups, peptidase labile groups, or esterase labile groups,as disclosed in the above-identified patents, disulfide and thioethergroups being preferred.

Conjugates of the antibody and maytansinoid may be made using a varietyof bifunctional protein coupling agents such asN-succinimidyl-3-(2-pyridyldithio) propionate (SPDP),succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate,iminothiolane (IT), bifunctional derivatives of imidoesters (such asdimethyl adipimidate HCL), active esters (such as disuccinimidylsuberate), aldehydes (such as glutareldehyde), bis-azido compounds (suchas bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (such asbis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astoluene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). Particularly preferred coupling agentsinclude N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP) (Carlssonet al., Biochem. J. 173:723-737 [1978]) andN-succinimidyl-4-(2-pyridylthio)pentanoate (SPP) to provide for adisulfide linkage.

The linker may be attached to the maytansinoid molecule at variouspositions, depending on the type of the link. For example, an esterlinkage may be formed by reaction with a hydroxyl group usingconventional coupling techniques. The reaction may occur at the C-3position having a hydroxyl group, the C-14 position modified withhydroxymethyl, the C-15 position modified with a hydroxyl group, and theC-20 position having a hydroxyl group. The linkage is formed at the C-3position of maytansinol or a maytansinol analogue.

Calicheamicin

Another immunoconjugate of interest comprises an anti-PRO226,anti-PRO257, anti-PRO268, anti-PRO290, anti-PRO36006, anti-PRO363,anti-PRO365, anti-PRO382, anti-PRO444, anti-PRO705, anti-PRO1071,anti-PRO1125, anti-PRO1134, anti-PRO1155, anti-PRO1281, anti-PRO1343,anti-PRO1379, anti-PRO1380, anti-PRO1387, anti-PRO1419, anti-PRO1433,anti-PRO1474, anti-PRO1550, anti-PRO1571, anti-PRO1572, anti-PRO1759,anti-PRO1904, anti-PRO35193, anti-PRO4341, anti-PRO4348, anti-PRO4369,anti-PRO4381, anti-PRO4407, anti-PRO4425, anti-PRO4985, anti-PRO4989,anti-PRO5737, anti-PRO5800, anti-PRO5993, anti-PRO6017, anti-PRO7174,anti-PRO9744, anti-PRO9821, anti-PRO9852, anti-PRO9873, anti-PRO10196,anti-PRO34778, anti-PRO20233, anti-PRO21956, anti-PRO57290,anti-PRO38465, anti-PRO38683 or anti-PRO85161 antibody conjugated to oneor more calicheamicin molecules. The calicheamicin family of antibioticsare capable of producing double-stranded DNA breaks at sub-picomolarconcentrations. For the preparation of conjugates of the calicheamicinfamily, see U.S. Pat. Nos. 5,712,374, 5,714,586, 5,739,116, 5,767,285,5,770,701, 5,770,710, 5,773,001, 5,877,296 (all to American CyanamidCompany). Structural analogues of calicheamicin which may be usedinclude, but are not limited to, γ₁ ¹, α₂ ¹, α₃ ¹, N-acetyl-γ₁ ¹, PSAGand θ¹ ₁ (Hinman et al., Cancer Research 53:3336-3342 (1993), Lode etal., Cancer Research 58:2925-2928 (1998) and the aforementioned U.S.patents to American Cyanamid). Another anti-tumor drug that the antibodycan be conjugated is QFA which is an antifolate. Both calicheamicin andQFA have intracellular sites of action and do not readily cross theplasma membrane. Therefore, cellular uptake of these agents throughantibody mediated internalization greatly enhances their cytotoxiceffects.

Other Cytotoxic Agents

Other antitumor agents that can be conjugated to the anti-PRO226,anti-PRO257, anti-PRO268, anti-PRO290, anti-PRO36006, anti-PRO363,anti-PRO365, anti-PRO382, anti-PRO444, anti-PRO705, anti-PRO1071,anti-PRO1125, anti-PRO1134, anti-PRO1155, anti-PRO1281, anti-PRO1343,anti-PRO1379, anti-PRO1380, anti-PRO1387, anti-PRO1419, anti-PRO1433,anti-PRO1474, anti-PRO1550, anti-PRO1571, anti-PRO1572, anti-PRO1759,anti-PRO1904, anti-PRO35193, anti-PRO4341, anti-PRO4348, anti-PRO4369,anti-PRO4381, anti-PRO4407, anti-PRO4425, anti-PRO4985, anti-PRO4989,anti-PRO5737, anti-PRO5800, anti-PRO5993, anti-PRO6017, anti-PRO7174,anti-PRO9744, anti-PRO9821, anti-PRO9852, anti-PRO9873, anti-PRO10196,anti-PRO34778, anti-PRO20233, anti-PRO21956, anti-PRO57290,anti-PRO38465, anti-PRO38683 or anti-PRO85161 antibodies of theinvention include BCNU, streptozoicin, vincristine and 5-fluorouracil,the family of agents known collectively LL-E33288 complex described inU.S. Pat. Nos. 5,053,394, 5,770,710, as well as esperamicins (U.S. Pat.No. 5,877,296).

Enzymatically active toxins and fragments thereof which can be usedinclude diphtheria A chain, nonbinding active fragments of diphtheriatoxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain,abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordiiproteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII,and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonariaofficinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin,enomycin and the tricothecenes. See, for example, WO 93/21232 publishedOct. 28, 1993.

The present invention further contemplates an immunoconjugate formedbetween an antibody and a compound with nucleolytic activity (e.g., aribonuclease or a DNA endonuclease such as a deoxyribonuclease; DNase).

For selective destruction of the tumor, the antibody may comprise ahighly radioactive atom. A variety of radioactive isotopes are availablefor the production of radioconjugated anti-PRO226, anti-PRO257,anti-PRO268, anti-PRO290, anti-PRO36006, anti-PRO363, anti-PRO365,anti-PRO382, anti-PRO444, anti-PRO705, anti-PRO1071, anti-PRO1125,anti-PRO1134, anti-PRO1155, anti-PRO1281, anti-PRO1343, anti-PRO1379,anti-PRO1380, anti-PRO1387, anti-PRO1419, anti-PRO1433, anti-PRO1474,anti-PRO1550, anti-PRO1571, anti-PRO1572, anti-PRO1759, anti-PRO1904,anti-PRO35193, anti-PRO4341, anti-PRO4348, anti-PRO4369, anti-PRO4381,anti-PRO4407, anti-PRO4425, anti-PRO4985, anti-PRO4989, anti-PRO5737,anti-PRO5800, anti-PRO5993, anti-PRO6017, anti-PRO7174, anti-PRO9744,anti-PRO9821, anti-PRO9852, anti-PRO9873, anti-PRO10196, anti-PRO34778,anti-PRO20233, anti-PRO21956, anti-PRO57290, anti-PRO38465,anti-PRO38683 or anti-PRO85161 antibodies. Examples include At²¹¹, I¹³¹,I¹²⁵, Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³, Bi²¹², P³², Pb²¹² and radioactiveisotopes of Lu. When the conjugate is used for diagnosis, it maycomprise a radioactive atom for scintigraphic studies, for exampletc^(99m) or I¹²³, or a spin label for nuclear magnetic resonance (NMR)imaging (also known as magnetic resonance imaging, mri), such asiodine-123 again, iodine-131, indium-111, fluorine-19, carbon-13,nitrogen-15, oxygen-17, gadolinium, manganese or iron.

The radio- or other labels may be incorporated in the conjugate in knownways. For example, the peptide may be biosynthesized or may besynthesized by chemical amino acid synthesis using suitable amino acidprecursors involving, for example, fluorine-19 in place of hydrogen.Labels such as tc^(99m) or I¹²³, Re¹⁸⁶, Re¹⁸⁸ and In¹¹¹ can be attachedvia a cysteine residue in the peptide. Yttrium-90 can be attached via alysine residue. The IODOGEN method (Fraker et al (1978) Biochem.Biophys. Res. Commun. 80: 49-57 can be used to incorporate iodine-123.“Monoclonal Antibodies in Immunoscintigraphy” (Chatal, CRC Press 1989)describes other methods in detail.

Conjugates of the antibody and cytotoxic agent may be made using avariety of bifunctional protein coupling agents such asN-succinimidyl-3-(2-pyridyldithio)propionate (SPDP),succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate,iminothiolane (IT), bifunctional derivatives of imidoesters (such asdimethyl adipimidate HCL), active esters (such as disuccinimidylsuberate), aldehydes (such as glutareldehyde), bis-azido compounds (suchas bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (such asbis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin canbe prepared as described in Vitetta et al., Science 238:1098 (1987).Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent forconjugation of radionucleotide to the antibody. See WO94/11026. Thelinker may be a “cleavable linker” facilitating release of the cytotoxicdrug in the cell. For example, an acid-labile linker,peptidase-sensitive linker, photolabile linker, dimethyl linker ordisulfide-containing linker (Chari et al., Cancer Research 52:127-131(1992); U.S. Pat. No. 5,208,020) may be used.

Alternatively, a fusion protein comprising the anti-PRO226, anti-PRO257,anti-PRO268, anti-PRO290, anti-PRO36006, anti-PRO363, anti-PRO365,anti-PRO382, anti-PRO444, anti-PRO705, anti-PRO1071, anti-PRO1125,anti-PRO1134, anti-PRO1155, anti-PRO1281, anti-PRO1343, anti-PRO1379,anti-PRO1380, anti-PRO1387, anti-PRO1419, anti-PRO1433, anti-PRO1474,anti-PRO1550, anti-PRO1571, anti-PRO1572, anti-PRO1759, anti-PRO1904,anti-PRO35193, anti-PRO4341, anti-PRO4348, anti-PRO4369, anti-PRO4381,anti-PRO4407, anti-PRO4425, anti-PRO4985, anti-PRO4989, anti-PRO5737,anti-PRO5800, anti-PRO5993, anti-PRO6017, anti-PRO7174, anti-PRO9744,anti-PRO9821, anti-PRO9852, anti-PRO9873, anti-PRO10196, anti-PRO34778,anti-PRO20233, anti-PRO21956, anti-PRO57290, anti-PRO38465,anti-PRO38683 or anti-PRO85161 antibody and cytotoxic agent may be made,e.g., by recombinant techniques or peptide synthesis. The length of DNAmay comprise respective regions encoding the two portions of theconjugate either adjacent one another or separated by a region encodinga linker peptide which does not destroy the desired properties of theconjugate.

The invention provides that the antibody may be conjugated to a“receptor” (such streptavidin) for utilization in tumor pre-targetingwherein the antibody-receptor conjugate is administered to the patient,followed by removal of unbound conjugate from the circulation using aclearing agent and then administration of a “ligand” (e.g., avidin)which is conjugated to a cytotoxic agent (e.g., a radionucleotide).

10. Immunoliposomes

The anti-PRO226, anti-PRO257, anti-PRO268, anti-PRO290, anti-PRO36006,anti-PRO363, anti-PRO365, anti-PRO382, anti-PRO444, anti-PRO705,anti-PRO1071, anti-PRO1125, anti-PRO1134, anti-PRO1155, anti-PRO1281,anti-PRO1343, anti-PRO1379, anti-PRO1380, anti-PRO1387, anti-PRO1419,anti-PRO1433, anti-PRO1474, anti-PRO1550, anti-PRO1571, anti-PRO1572,anti-PRO1759, anti-PRO1904, anti-PRO35193, anti-PRO4341, anti-PRO4348,anti-PRO4369, anti-PRO4381, anti-PRO4407, anti-PRO4425, anti-PRO4985,anti-PRO4989, anti-PRO5737, anti-PRO5800, anti-PRO5993, anti-PRO6017,anti-PRO7174, anti-PRO9744, anti-PRO9821, anti-PRO9852, anti-PRO9873,anti-PRO10196, anti-PRO34778, anti-PRO20233, anti-PRO21956,anti-PRO57290, anti-PRO38465, anti-PRO38683 or anti-PRO85161 antibodiesdisclosed herein may also be formulated as immunoliposomes. A “liposome”is a small vesicle composed of various types of lipids, phospholipidsand/or surfactant which is useful for delivery of a drug to a mammal.The components of the liposome are commonly arranged in a bilayerformation, similar to the lipid arrangement of biological membranes.Liposomes containing the antibody are prepared by methods known in theart, such as described in Epstein et al., Proc. Natl. Acad. Sci. USA82:3688 (1985); Hwang et al., Proc. Natl. Acad. Sci. USA 77:4030 (1980);U.S. Pat. Nos. 4,485,045 and 4,544,545; and WO97/38731 published Oct.23, 1997. Liposomes with enhanced circulation time are disclosed in U.S.Pat. No. 5,013,556.

Particularly useful liposomes can be generated by the reverse phaseevaporation method with a lipid composition comprisingphosphatidylcholine, cholesterol and PEG-derivatizedphosphatidylethanolamine (PEG-PE). Liposomes are extruded throughfilters of defined pore size to yield liposomes with the desireddiameter. Fab′ fragments of the antibody of the present invention can beconjugated to the liposomes as described in Martin et al., J. Biol.Chem. 257:286-288 (1982) via a disulfide interchange reaction. Achemotherapeutic agent is optionally contained within the liposome. SeeGabizon et al., J. National Cancer Inst. 81(19):1484 (1989).

11. Pharmaceutical Compositions of Antibodies

Antibodies specifically binding a PRO226, PRO257, PRO268, PRO290,PRO36006, PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125,PRO1134, PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419,PRO1433, PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193,PRO4341, PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989,PRO5737, PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852,PRO9873, PRO10196, PRO34778, PRO20233, PRO21956, PRO57290, PRO38465,PRO38683 or PRO85161 polypeptide identified herein, as well as othermolecules identified by the screening assays disclosed hereinbefore, canbe administered for the treatment of various disorders in the form ofpharmaceutical compositions.

If the PRO226, PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382,PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343,PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571,PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381,PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017,PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196, PRO34778,PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161 polypeptideis intracellular and whole antibodies are used as inhibitors,internalizing antibodies are preferred. However, lipofections orliposomes can also be used to deliver the antibody, or an antibodyfragment, into cells. Where antibody fragments are used, the smallestinhibitory fragment that specifically binds to the binding domain of thetarget protein is preferred. For example, based upon the variable-regionsequences of an antibody, peptide molecules can be designed that retainthe ability to bind the target protein sequence. Such peptides can besynthesized chemically and/or produced by recombinant DNA technology.See, e.g., Marasco et al., Proc. Natl. Acad. Sci. USA, 90: 7889-7893(1993). The formulation herein may also contain more than one activecompound as necessary for the particular indication being treated,preferably those with complementary activities that do not adverselyaffect each other. Alternatively, or in addition, the composition maycomprise an agent that enhances its function, such as, for example, acytotoxic agent, cytokine, chemotherapeutic agent, or growth-inhibitoryagent. Such molecules are suitably present in combination in amountsthat are effective for the purpose intended.

The active ingredients may also be entrapped in microcapsules prepared,for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsules and poly-(methylmethacylate) microcapsules,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles, andnanocapsules) or in macroemulsions. Such techniques are disclosed inRemington's Pharmaceutical Sciences, supra.

The formulations to be used for in vivo administration must be sterile.This is readily accomplished by filtration through sterile filtrationmembranes.

Sustained-release preparations may be prepared. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymers containing the antibody, which matrices are in theform of shaped articles, e.g., films, or microcapsules. Examples ofsustained-release matrices include polyesters, hydrogels (for example,poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides(U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and γethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradablelactic acid-glycolic acid copolymers such as the LUPRON DEPOT™(injectable microspheres composed of lactic acid-glycolic acid copolymerand leuprolide acetate), and poly-D-(−)-3-hydroxybutyric acid.

While polymers such as ethylene-vinyl acetate and lactic acid-glycolicacid enable release of molecules for over 100 days, certain hydrogelsrelease proteins for shorter time periods. When encapsulated antibodiesremain in the body for a long time, they may denature or aggregate as aresult of exposure to moisture at 37° C., resulting in a loss ofbiological activity and possible changes in immunogenicity. Rationalstrategies can be devised for stabilization depending on the mechanisminvolved. For example, if the aggregation mechanism is discovered to beintermolecular S—S bond formation through thio-disulfide interchange,stabilization may be achieved by modifying sulfhydryl residues,lyophilizing from acidic solutions, controlling moisture content, usingappropriate additives, and developing specific polymer matrixcompositions.

G. Uses for Anti-PRO226, Anti-PRO257, Anti-PRO268, Anti-PRO290,Anti-PRO36006, Anti-PRO363, Anti-PRO365, Anti-PRO382, Anti-PRO444,Anti-PRO705, Anti-PRO1071, Anti-PRO1125, Anti-PRO1134, Anti-PRO1155,Anti-PRO1281, Anti-PRO1343, Anti-PRO1379, Anti-PRO1380, Anti-PRO1387,Anti-PRO1419, Anti-PRO1433, Anti-PRO1474, Anti-PRO1550, Anti-PRO1571,Anti-PRO1572, Anti-PRO1759, Anti-PRO1904, Anti-PRO35193, Anti-PRO4341,Anti-PRO4348, Anti-PRO4369, Anti-PRO4381, Anti-PRO4407, Anti-PRO4425,Anti-PRO4985, Anti-PRO4989, Anti-PRO5737, Anti-PRO5800, Anti-PRO5993,Anti-PRO6017, Anti-PRO7174, Anti-PRO9744, Anti-PRO9821, Anti-PRO9852,Anti-PRO9873, Anti-PRO10196, Anti-PRO34778, Anti-PRO20233,Anti-PRO21956, Anti-PRO57290, Anti-PRO38465, Anti-PRO38683 orAnti-PRO85161 Antibodies

The anti-PRO226, anti-PRO257, anti-PRO268, anti-PRO290, anti-PRO36006,anti-PRO363, anti-PRO365, anti-PRO382, anti-PRO444, anti-PRO705,anti-PRO1071, anti-PRO1125, anti-PRO1134, anti-PRO1155, anti-PRO1281,anti-PRO1343, anti-PRO1379, anti-PRO1380, anti-PRO1387, anti-PRO1419,anti-PRO1433, anti-PRO1474, anti-PRO1550, anti-PRO1571, anti-PRO1572,anti-PRO1759, anti-PRO1904, anti-PRO35193, anti-PRO4341, anti-PRO4348,anti-PRO4369, anti-PRO4381, anti-PRO4407, anti-PRO4425, anti-PRO4985,anti-PRO4989, anti-PRO5737, anti-PRO5800, anti-PRO5993, anti-PRO6017,anti-PRO7174, anti-PRO9744, anti-PRO9821, anti-PRO9852, anti-PRO9873,anti-PRO10196, anti-PRO34778, anti-PRO20233, anti-PRO21956,anti-PRO57290, anti-PRO38465, anti-PRO38683 or anti-PRO85161 antibodiesof the invention have various therapeutic and/or diagnostic utilitiesfor a neurological disorder; a cardiovascular, endothelial or angiogenicdisorder; an immunological disorder; an oncological disorder; anembryonic developmental disorder or lethality, or a metabolicabnormality. For example, anti-PRO226, anti-PRO257, anti-PRO268,anti-PRO290, anti-PRO36006, anti-PRO363, anti-PRO365, anti-PRO382,anti-PRO444, anti-PRO705, anti-PRO1071, anti-PRO1125, anti-PRO1134,anti-PRO1155, anti-PRO1281, anti-PRO1343, anti-PRO1379, anti-PRO1380,anti-PRO1387, anti-PRO1419, anti-PRO1433, anti-PRO1474, anti-PRO1550,anti-PRO1571, anti-PRO1572, anti-PRO1759, anti-PRO1904, anti-PRO35193,anti-PRO4341, anti-PRO4348, anti-PRO4369, anti-PRO4381, anti-PRO4407,anti-PRO4425, anti-PRO4985, anti-PRO4989, anti-PRO5737, anti-PRO5800,anti-PRO5993, anti-PRO6017, anti-PRO7174, anti-PRO9744, anti-PRO9821,anti-PRO9852, anti-PRO9873, anti-PRO10196, anti-PRO34778, anti-PRO20233,anti-PRO21956, anti-PRO57290, anti-PRO38465, anti-PRO38683 oranti-PRO85161 antibodies may be used in diagnostic assays for PRO226,PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382, PRO444,PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343, PRO1379,PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571, PRO1572,PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381, PRO4407,PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017, PRO7174,PRO9744, PRO9821, PRO9852, PRO9873, PRO10196, PRO34778, PRO20233,PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161, e.g., detecting itsexpression (and in some cases, differential expression) in specificcells, tissues, or serum. Various diagnostic assay techniques known inthe art may be used, such as competitive binding assays, direct orindirect sandwich assays and immunoprecipitation assays conducted ineither heterogeneous or homogeneous phases [Zola, Monoclonal Antibodies:A Manual of Techniques, CRC Press, Inc. (1987) pp. 147-158]. Theantibodies used in the diagnostic assays can be labeled with adetectable moiety. The detectable moiety should be capable of producing,either directly or indirectly, a detectable signal. For example, thedetectable moiety may be a radioisotope, such as ³H, ¹⁴C, ³²P, ³⁵S, or¹²⁵I, a fluorescent or chemiluminescent compound, such as fluoresceinisothiocyanate, rhodamine, or luciferin, or an enzyme, such as alkalinephosphatase, beta-galactosidase or horseradish peroxidase. Any methodknown in the art for conjugating the antibody to the detectable moietymay be employed, including those methods described by Hunter et al.,Nature, 144:945 (1962); David et al., Biochemistry, 13: 1014 (1974);Pain et al., J. Immunol. Meth., 40:219 (1981); and Nygren, J. Histochem.and Cytochem., 30:407 (1982).

Anti-PRO226, anti-PRO257, anti-PRO268, anti-PRO290, anti-PRO36006,anti-PRO363, anti-PRO365, anti-PRO382, anti-PRO444, anti-PRO705,anti-PRO1071, anti-PRO1125, anti-PRO1134, anti-PRO1155, anti-PRO1281,anti-PRO1343, anti-PRO1379, anti-PRO1380, anti-PRO1387, anti-PRO1419,anti-PRO1433, anti-PRO1474, anti-PRO1550, anti-PRO1571, anti-PRO1572,anti-PRO1759, anti-PRO1904, anti-PRO35193, anti-PRO4341, anti-PRO4348,anti-PRO4369, anti-PRO4381, anti-PRO4407, anti-PRO4425, anti-PRO4985,anti-PRO4989, anti-PRO5737, anti-PRO5800, anti-PRO5993, anti-PRO6017,anti-PRO7174, anti-PRO9744, anti-PRO9821, anti-PRO9852, anti-PRO9873,anti-PRO10196, anti-PRO34778, anti-PRO20233, anti-PRO21956,anti-PRO57290, anti-PRO38465, anti-PRO38683 or anti-PRO85161 antibodiesalso are useful for the affinity purification of PRO226, PRO257, PRO268,PRO290, PRO36006, PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071,PRO1125, PRO1134, PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387,PRO1419, PRO1433, PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904,PRO35193, PRO4341, PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985,PRO4989, PRO5737, PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821,PRO9852, PRO9873, PRO10196, PRO34778, PRO20233, PRO21956, PRO57290,PRO38465, PRO38683 or PRO85161 polypeptides from recombinant cellculture or natural sources. In this process, the antibodies againstPRO226, PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382,PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343,PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571,PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381,PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017,PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196, PRO34778,PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161polypeptides are immobilized on a suitable support, such a Sephadexresin or filter paper, using methods well known in the art. Theimmobilized antibody then is contacted with a sample containing thePRO226, PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382,PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343,PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571,PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381,PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017,PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196, PRO34778,PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161 polypeptideto be purified, and thereafter the support is washed with a suitablesolvent that will remove substantially all the material in the sampleexcept the PRO226, PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365,PRO382, PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281,PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550,PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369,PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993,PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196,PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161polypeptide, which is bound to the immobilized antibody. Finally, thesupport is washed with another suitable solvent that will release thePRO226, PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382,PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343,PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571,PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381,PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017,PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196, PRO34778,PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161 polypeptidefrom the antibody.

The following examples are offered for illustrative purposes only, andare not intended to limit the scope of the present invention in any way.

All patent and literature references cited in the present specificationare hereby incorporated by reference in their entirety.

EXAMPLES

Commercially available reagents referred to in the examples were usedaccording to manufacturer's instructions unless otherwise indicated. Thesource of those cells identified in the following examples, andthroughout the specification, by ATCC accession numbers is the AmericanType Culture Collection, Manassas, Va.

Example 1 Extracellular Domain Homology Screening to Identify NovelPolypeptides and cDNA Encoding Therefor

The extracellular domain (ECD) sequences (including the secretion signalsequence, if any) from about 950 known secreted proteins from theSwiss-Prot public database were used to search EST databases. The ESTdatabases included public databases (e.g., Dayhoff, GenBank), andproprietary databases (e.g. LIFESEQ™, Incyte Pharmaceuticals, Palo Alto,Calif.). The search was performed using the computer program BLAST orBLAST-2 (Altschul et al., Methods in Enzymology, 266:460-480 (1996)) asa comparison of the ECD protein sequences to a 6 frame translation ofthe EST sequences. Those comparisons with a BLAST score of 70 (or insome cases 90) or greater that did not encode known proteins wereclustered and assembled into consensus DNA sequences with the program“phrap” (Phil Green, University of Washington, Seattle, Wash.).

Using this extracellular domain homology screen, consensus DNA sequenceswere assembled relative to the other identified EST sequences usingphrap. In addition, the consensus DNA sequences obtained were often (butnot always) extended using repeated cycles of BLAST or BLAST-2 and phrapto extend the consensus sequence as far as possible using the sources ofEST sequences discussed above.

Based upon the consensus sequences obtained as described above,oligonucleotides were then synthesized and used to identify by PCR acDNA library that contained the sequence of interest and for use asprobes to isolate a clone of the full-length coding sequence for a PROpolypeptide. Forward and reverse PCR primers generally range from 20 to30 nucleotides and are often designed to give a PCR product of about100-1000 bp in length. The probe sequences are typically 40-55 bp inlength. In some cases, additional oligonucleotides are synthesized whenthe consensus sequence is greater than about 1-1.5 kbp. In order toscreen several libraries for a full-length clone, DNA from the librarieswas screened by PCR amplification, as per Ausubel et al., CurrentProtocols in Molecular Biology, with the PCR primer pair. A positivelibrary was then used to isolate clones encoding the gene of interestusing the probe oligonucleotide and one of the primer pairs.

The cDNA libraries used to isolate the cDNA clones were constructed bystandard methods using commercially available reagents such as thosefrom Invitrogen, San Diego, Calif. The cDNA was primed with oligo dTcontaining a NotI site, linked with blunt to SalI hemikinased adaptors,cleaved with NotI, sized appropriately by gel electrophoresis, andcloned in a defined orientation into a suitable cloning vector (such aspRKB or pRKD; pRK5B is a precursor of pRK5D that does not contain theSfiI site; see, Holmes et al., Science, 253:1278-1280 (1991)) in theunique XhoI and NotI sites.

Example 2 Isolation of cDNA Clones by Amylase Screening

1. Preparation of Oligo dT Primed cDNA Library

mRNA was isolated from a human tissue of interest using reagents andprotocols from Invitrogen, San Diego, Calif. (Fast Track 2). This RNAwas used to generate an oligo dT primed cDNA library in the vector pRK5Dusing reagents and protocols from Life Technologies, Gaithersburg, Md.(Super Script Plasmid System). In this procedure, the double strandedcDNA was sized to greater than 1000 bp and the SalI/NotI linkered cDNAwas cloned into XhoI/NotI cleaved vector. pRK5D is a cloning vector thathas an sp6 transcription initiation site followed by an SfiI restrictionenzyme site preceding the XhoI/NotI cDNA cloning sites.

2. Preparation of Random Primed cDNA Library

A secondary cDNA library was generated in order to preferentiallyrepresent the 5′ ends of the primary cDNA clones. Sp6 RNA was generatedfrom the primary library (described above), and this RNA was used togenerate a random primed cDNA library in the vector pSST-AMY.0 usingreagents and protocols from Life Technologies (Super Script PlasmidSystem, referenced above). In this procedure the double stranded cDNAwas sized to 500-1000 bp, linkered with blunt to NotI adaptors, cleavedwith SfiI, and cloned into SfiI/NotI cleaved vector. pSST-AMY.0 is acloning vector that has a yeast alcohol dehydrogenase promoter precedingthe cDNA cloning sites and the mouse amylase sequence (the maturesequence without the secretion signal) followed by the yeast alcoholdehydrogenase terminator, after the cloning sites. Thus, cDNAs clonedinto this vector that are fused in frame with amylase sequence will leadto the secretion of amylase from appropriately transfected yeastcolonies.

3. Transformation and Detection

DNA from the library described in paragraph 2 above was chilled on iceto which was added electrocompetent DH10B bacteria (Life Technologies,20 ml). The bacteria and vector mixture was then electroporated asrecommended by the manufacturer. Subsequently, SOC media (LifeTechnologies, 1 ml) was added and the mixture was incubated at 37° C.for 30 minutes. The transformants were then plated onto 20 standard 150mm LB plates containing ampicillin and incubated for 16 hours (37° C.).Positive colonies were scraped off the plates and the DNA was isolatedfrom the bacterial pellet using standard protocols, e.g. CsCl-gradient.The purified DNA was then carried on to the yeast protocols below.

The yeast methods were divided into three categories: (1) Transformationof yeast with the plasmid/cDNA combined vector; (2) Detection andisolation of yeast clones secreting amylase; and (3) PCR amplificationof the insert directly from the yeast colony and purification of the DNAfor sequencing and further analysis.

The yeast strain used was HD56-5A (ATCC-90785). This strain has thefollowing genotype: MAT alpha, ura3-52, leu2-3, leu2-112, his3-11,his3-15, MAL⁺, SUC⁺, GAL⁺. Preferably, yeast mutants can be employedthat have deficient post-translational pathways. Such mutants may havetranslocation deficient alleles in sec71, sec72, sec62, with truncatedsec71 being most preferred. Alternatively, antagonists (includingantisense nucleotides and/or ligands) which interfere with the normaloperation of these genes, other proteins implicated in this posttranslation pathway (e.g., SEC61p, SEC72p, SEC62p, SEC63p, TDJ1p orSSA1p-4-p) or the complex formation of these proteins may also bepreferably employed in combination with the amylase-expressing yeast.

Transformation was performed based on the protocol outlined by Gietz etal., Nucl. Acid. Res., 20:1425 (1992). Transformed cells were theninoculated from agar into YEPD complex media broth (100 ml) and grownovernight at 30° C. The YEPD broth was prepared as described in Kaiseret al., Methods in Yeast Genetics, Cold Spring Harbor Press, Cold SpringHarbor, N.Y., p. 207 (1994). The overnight culture was then diluted toabout 2×10⁶ cells/ml (approx. OD₆₀₀=0.1) into fresh YEPD broth (500 ml)and regrown to 1×10⁷ cells/ml (approx. OD₆₀₀=0.4-0.5).

The cells were then harvested and prepared for transformation bytransfer into GS3 rotor bottles in a Sorval GS3 rotor at 5,000 rpm for 5minutes, the supernatant discarded, and then resuspended into sterilewater, and centrifuged again in 50 ml falcon tubes at 3,500 rpm in aBeckman GS-6KR centrifuge. The supernatant was discarded and the cellswere subsequently washed with LiAc/TE (10 ml, 10 mM Tris-HCl, 1 mM EDTApH 7.5, 100 mM Li₂OOCCH₃), and resuspended into LiAc/TE (2.5 ml).

Transformation took place by mixing the prepared cells (100 μl) withfreshly denatured single stranded salmon testes DNA (Lofstrand Labs,Gaithersburg, Md.) and transforming DNA (1 μg, vol. <10 μl) in microfugetubes. The mixture was mixed briefly by vortexing, then 40% PEG/TE (600μl, 40% polyethylene glycol-4000, mM Tris-HCl, 1 mM EDTA, 100 mMLi₂OOCCH₃, pH 7.5) was added. This mixture was gently mixed andincubated at 30° C. while agitating for 30 minutes. The cells were thenheat shocked at 42° C. for 15 minutes, and the reaction vesselcentrifuged in a microfuge at 12,000 rpm for 5-10 seconds, decanted andresuspended into TE (500 μl, 10 mM Tris-HCl, 1 mM EDTA pH 7.5) followedby recentrifugation. The cells were then diluted into TE (1 ml) andaliquots (200 μl) were spread onto the selective media previouslyprepared in 150 mm growth plates (VWR).

Alternatively, instead of multiple small reactions, the transformationwas performed using a single, large scale reaction, wherein reagentamounts were scaled up accordingly.

The selective media used was a synthetic complete dextrose agar lackinguracil (SCD-Ura) prepared as described in Kaiser et al., Methods inYeast Genetics, Cold Spring Harbor Press, Cold Spring Harbor, N.Y., p.208-210 (1994). Transformants were grown at 30° C. for 2-3 days.

The detection of colonies secreting amylase was performed by includingred starch in the selective growth media. Starch was coupled to the reddye (Reactive Red-120, Sigma) as per the procedure described by Biely etal., Anal. Biochem., 172:176-179 (1988). The coupled starch wasincorporated into the SCD-Ura agar plates at a final concentration of0.15% (w/v), and was buffered with potassium phosphate to a pH of 7.0(50-100 mM final concentration).

The positive colonies were picked and streaked across fresh selectivemedia (onto 150 mm plates) in order to obtain well isolated andidentifiable single colonies. Well isolated single colonies positive foramylase secretion were detected by direct incorporation of red starchinto buffered SCD-Ura agar. Positive colonies were determined by theirability to break down starch resulting in a clear halo around thepositive colony visualized directly.

4. Isolation of DNA by PCR Amplification

When a positive colony was isolated, a portion of it was picked by atoothpick and diluted into sterile water (30 μl) in a 96 well plate. Atthis time, the positive colonies were either frozen and stored forsubsequent analysis or immediately amplified. An aliquot of cells (5 μl)was used as a template for the PCR reaction in a 25 μl volumecontaining: 0.5 μl Klentaq (Clontech, Palo Alto, Calif.); 4.0 μl 10 mMdNTP's (Perkin Elmer-Cetus); 2.5 μl Kentaq buffer (Clontech); 0.25 μlforward oligo 1; 0.25 μl reverse oligo 2; 12.5 μl distilled water. Thesequence of the forward oligonucleotide 1 was:

(SEQ ID NO: 107) 5′-TGTAAAACGACGGCCAGTTAAATAGACCTGCAATTATTAATCT-3′The sequence of reverse of oligonucleotide 2 was:

(SEQ ID NO: 108) 5′-CAGGAAACAGCTATGACCACCTGCACACCTGCAAATCCATT-3′PCR was then performed as follows:

a. Denature 92° C.,  5 minutes b.  3 cycles of: Denature 92° C., 30seconds Anneal 59° C., 30 seconds Extend 72° C., 60 seconds c.  3 cyclesof: Denature 92° C., 30 seconds Anneal 57° C., 30 seconds Extend 72° C.,60 seconds d. 25 cycles of: Denature 92° C., 30 seconds Anneal 55° C.,30 seconds Extend 72° C., 60 seconds e. Hold  4° C.

The underlined regions of the oligonucleotides annealed to the ADHpromoter region and the amylase region, respectively, and amplified a307 bp region from vector pSST-AMY.0 when no insert was present.Typically, the first 18 nucleotides of the 5′ end of theseoligonucleotides contained annealing sites for the sequencing primers.Thus, the total product of the PCR reaction from an empty vector was 343bp. However, signal sequence-fused cDNA resulted in considerably longernucleotide sequences.

Following the PCR, an aliquot of the reaction (5 μl) was examined byagarose gel electrophoresis in a 1% agarose gel using a Tris-Borate-EDTA(TBE) buffering system as described by Sambrook et al., supra. Clonesresulting in a single strong PCR product larger than 400 bp were furtheranalyzed by DNA sequencing after purification with a 96 Qiaquick PCRclean-up column (Qiagen Inc., Chatsworth, Calif.).

Example 3 Isolation of cDNA Clones Using Signal Algorithm Analysis

Various polypeptide-encoding nucleic acid sequences were identified byapplying a proprietary signal sequence finding algorithm developed byGenentech, Inc. (South San Francisco, Calif.) upon ESTs as well asclustered and assembled EST fragments from public (e.g., GenBank) and/orprivate (LIFESEQ®, Incyte Pharmaceuticals, Inc., Palo Alto, Calif.)databases. The signal sequence algorithm computes a secretion signalscore based on the character of the DNA nucleotides surrounding thefirst and optionally the second methionine codon(s) (ATG) at the 5′-endof the sequence or sequence fragment under consideration. Thenucleotides following the first ATG must code for at least 35unambiguous amino acids without any stop codons. If the first ATG hasthe required amino acids, the second is not examined. If neither meetsthe requirement, the candidate sequence is not scored. In order todetermine whether the EST sequence contains an authentic signalsequence, the DNA and corresponding amino acid sequences surrounding theATG codon are scored using a set of seven sensors (evaluationparameters) known to be associated with secretion signals. Use of thisalgorithm resulted in the identification of numerouspolypeptide-encoding nucleic acid sequences.

Using the techniques described in Examples 1 to 3 above, numerousfull-length cDNA clones were identified as encoding PRO226, PRO257,PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382, PRO444, PRO705,PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343, PRO1379, PRO1380,PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571, PRO1572, PRO1759,PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381, PRO4407, PRO4425,PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017, PRO7174, PRO9744,PRO9821, PRO9852, PRO9873, PRO10196, PRO34778, PRO20233, PRO21956,PRO57290, PRO38465, PRO38683 or PRO85161 polypeptides as disclosedherein. These cDNAs were then deposited under the terms of the BudapestTreaty with the American Type Culture Collection, 10801 UniversityBlvd., Manassas, Va. 20110-2209, USA (ATCC) as shown in Table 7 below.In addition, the sequence of DNA225543 encoding PRO36006 polypeptideswas identified from GenBank accession no.: AF170484; the sequence ofDNA82372 encoding PRO1904 polypeptides was identified from GenBankaccession no.: AB007454; the sequence of DNA225681 encoding PRO35193polypeptides was identified from GenBank accession no.: D14012; thesequence of DNA220432 encoding PRO34778 polypeptides was identified fromGenBank accession no.: AF369708; the sequence of DNA165608 encodingPRO20233 polypeptides was identified from GenBank accession no.:AF286095; the sequence of DNA269238 encoding PRO57290 polypeptides wasidentified from GenBank accession no.: AF326591; the sequence ofDNA228002 encoding PRO38465 polypeptides was identified from GenBankaccession no.: AF412-409; the sequence of DNA228199 encoding PRO38683polypeptides was identified from GenBank accession no.: AK024365; andthe sequence of DNA329632 encoding PRO85161 polypeptides was identifiedfrom GenBank accession no.: AF479260.

TABLE 7 Material ATCC Dep. No. Deposit Date DNA33460-1166 209376 Oct.16, 1997 DNA35841-1173 209403 Oct. 17, 1997 DNA39427-1179 209395 Oct.17, 1997 DNA35680-1212 209790 Apr. 21, 1998 DNA45419-1252 209616 Feb. 5,1998 DNA46777-1253 209619 Feb. 5, 1998 DNA45234-1277 209654 Mar. 5, 1998DNA26846-1397 203406 Oct. 27, 1998 DNA50914-1289 209722 Mar. 31, 1998DNA58847-1383 209879 May 20, 1998 DNA60619-1482 209993 Jun. 16, 1998DNA56865-1491 203022 Jun. 23, 1998 DNA59849-1504 209986 Jun. 16, 1998DNA59820-1549 203129 Aug. 18, 1998 DNA66675-1587 203282 Sep. 22, 1998DNA59828-1608 203158 Aug. 25, 1998 DNA60740-1615 203456 Nov. 3, 1998DNA68872-1620 203160 Aug. 25, 1998 DNA71290-1630 203275 Sep. 22, 1998DNA71184-1634 203266 Sep. 22, 1998 DNA73739-1645 203270 Sep. 22, 1998DNA76393-1664 203323 Oct. 6, 1998 DNA73730-1679 203320 Oct. 6, 1998DNA73734-1680 203363 Oct. 20, 1998 DNA76531-1701 203465 Nov. 17, 1998DNA81761-2583 203862 Mar. 23, 1999 DNA92232-2589 203895 Mar. 30, 1999DNA92289-2598 PTA-131 May 25, 1999 DNA92225-2603 203950 Apr. 20, 1999DNA92264-2616 203969 Apr. 27, 1999 DNA93011-2637 PTA-20 May 4, 1999DNA59770-2652 PTA-427 Jul. 27, 1999 DNA80135-2655 PTA-234 Jun. 15, 1999DNA92929-2534-1 203586 Jan. 12, 1999 DNA108912-2680 PTA-124 May 25, 1999DNA100276-2684 PTA-380 Jul. 20, 1999 DNA96860-2700 PTA-478 Aug. 3, 1999DNA96883-2745 PTA-544 Aug. 17, 1999 DNA136110-2763 PTA-652 Sep. 14, 1999DNA108725-2766 PTA-863 Oct. 19, 1999 DNA129332-2775 PTA-944 Nov. 9, 1999DNA143076-2787 PTA-1028 Dec. 7, 1999 DNA144841-2816 PTA-1188 Jan. 11,2000 DNA178511-2986 PTA-2452 Sep. 12, 2000

These deposits were made under the provisions of the Budapest Treaty onthe International Recognition of the Deposit of Microorganisms for thePurpose of Patent Procedure and the Regulations thereunder (BudapestTreaty). This assures maintenance of a viable culture of the deposit for30 years from the date of deposit. The deposits will be made availableby ATCC under the terms of the Budapest Treaty, and subject to anagreement between Genentech, Inc. and ATCC, which assures permanent andunrestricted availability of the progeny of the culture of the depositto the public upon issuance of the pertinent U.S. patent or upon layingopen to the public of any U.S. or foreign patent application, whichevercomes first, and assures availability of the progeny to one determinedby the U.S. Commissioner of Patents and Trademarks to be entitledthereto according to 35 USC §122 and the Commissioner's rules pursuantthereto (including 37 CFR §1.14 with particular reference to 8860G 638).

The assignee of the present application has agreed that if a culture ofthe materials on deposit should die or be lost or destroyed whencultivated under suitable conditions, the materials will be promptlyreplaced on notification with another of the same. Availability of thedeposited material is not to be construed as a license to practice theinvention in contravention of the rights granted under the authority ofany government in accordance with its patent laws.

Example 4 Isolation of cDNA Clones Encoding Human PRO226 Polypeptides[UNQ200]

A consensus DNA sequence was assembled relative to other EST sequencesusing phrap as described in Example 1 above. This assembled consensussequence encoding an EGF-like homologue is herein identified asDNA28744. Based on the DNA28744 consensus sequence, oligonucleotideswere synthesized: 1) to identify by PCR a cDNA library that containedthe sequence of interest, and 2) for use as probes to isolate a clone ofthe full-length coding sequence for PRO226.

PCR primers (forward and reverse) were synthesized:

forward PCR primer (28744.f) (OLI556): 5′-ATTCTGCGTGAACACTGAGGGC-3′(SEQ ID NO: 109) reverse PCR primer (28744.r) (OLI557):5′-ATCTGCTTGTAGCCCTCGGCAC-3′ (SEQ ID NO: 110)Additionally, a synthetic oligonucleotide hybridization probe wasconstructed from the DNA28744 consensus sequence which had the followingnucleotide sequence:

hybridization probe (28744.p) (OLI555): (SEQ ID NO: 111)5′-CCTGGCTATCAGCAGGTGGGCTCCAAGTGTCTCGATGTGGATGAGTG TGA-3′

In order to screen several libraries for a source of a full-lengthclone, DNA from the libraries was screened by PCR amplification with thePCR primer pairs identified above. A positive library was then used toisolate clones encoding the PRO226 gene using the probe oligonucleotideand one of the PCR primers. RNA for construction of the cDNA librarieswas isolated from human fetal lung tissue.

DNA sequencing of the isolated clones isolated as described above gavethe full-length DNA sequence for DNA33460-1166 [FIG. 1, SEQ ID NO:1];and the derived protein sequence for PRO226.

The entire coding sequence of DNA33460-1166 is included in FIG. 1 (SEQID NO: 1). Clone DNA33460-1166 contains a single open reading frame withan apparent translational initiation site at nucleotide positions 62-64,and an apparent stop codon at nucleotide positions 1391-1393. Thepredicted polypeptide precursor is 443 amino acids long. Analysis of thefull-length PRO226 sequence shown in FIG. 2 (SEQ ID NO:2) evidences thepresence of a variety of important polypeptide domains, wherein thelocations given for those important polypeptide domains are approximateas described above. Analysis of the full-length PRO226 polypeptide shownin FIG. 2 evidences the presence of the following: a signal peptide fromabout amino acid 1 to about amino acid 25; N-glycosylation sites fromabout amino acid 198 to about amino acid 202 and from about amino acid394 to about amino acid 398; N-myristoylation sites from about aminoacid 76 to about amino acid 82, from about amino acid 145 to about aminoacid 151, from about amino acid 182 to about amino acid 188, from aboutamino acid 222 to about amino acid 228, from about amino acid 290 toabout amino acid 296, from about amino acid 305 to about amino acid 311,from about amino acid 371 to about amino acid 377 and from about aminoacid 381 to about amino acid 387; and aspartic acid and asparaginehydroxylation sites from about amino acid 140 to about amino acid 152,from about amino acid 177 to about amino acid 189, from about amino acid217 to about amino acid 229, and from about amino acid 258 to aboutamino acid 270. Clone DNA33460-1166 has been deposited with the ATCC onOct. 16, 1997 and is assigned ATCC deposit no. 209376.

Based on a BLAST and FastA sequence alignment analysis of thefull-length PRO226 sequence shown in FIG. 2 (SEQ ID NO:2), EGF-likehomolog DNA33460-1166 shows amino acid sequence identity to HT proteinand/or Fibulin (49% and 38%, respectively).

Example 5 Isolation of cDNA Clones Encoding Human PRO257 Polypeptides[UNQ224]

A consensus DNA sequence was assembled relative to other EST sequencesusing phrap as described in Example 1 above. This consensus sequence isherein designated DNA28731. Based on the DNA28731 consensus sequence,oligonucleotides were synthesized: 1) to identify by PCR a cDNA librarythat contained the sequence of interest, and 2) for use as probes toisolate a clone of the full-length coding sequence for PRO257.

A pair of PCR primers (forward and reverse) were synthesized:

forward PCR primer 5′-TCTCTATTCCAAACTGTGGCG-3′ (SEQ ID NO: 112)reverse PCR primer 5′-TTTGATGACGATTCGAAGGTGG-3′ (SEQ ID NO: 113)Additionally, a synthetic oligonucleotide hybridization probe wasconstructed from the consensus DNA28731 sequence which had the followingnucleotide sequence hybridization probe

hybridization probe (SEQ ID NO: 114)5′-GGAAGGATCCTTCACCAGCCCCAATTACCCAAAGCCGCATCCTGAG C-3′

In order to screen several libraries for a source of a full-lengthclone, DNA from the libraries was screened by PCR amplification with thePCR primer pair identified above. A positive library was then used toisolate clones encoding the PRO257 gene using the probe oligonucleotideand one of the PCR primers.

RNA for construction of the cDNA libraries was isolated from human fetalkidney tissue.

DNA sequencing of the clones isolated as described above gave thefull-length DNA sequence for PRO257 [herein designated as DNA35841-1173(FIG. 3; SEQ ID NO:3) and the derived protein sequence for PRO257.

The entire nucleotide sequence of DNA35841-1173 is shown in FIG. 3 (SEQID NO:3). Clone DNA35841-1173 contains a single open reading frame withan apparent translational initiation site at nucleotide positions964-966 and ending at the stop codon at nucleotide positions 2785-2787(FIG. 3). The predicted polypeptide precursor is 607 amino acids long(FIG. 4; SEQ ID NO:4). Clone DNA35841-1173 has been deposited with ATCCon Oct. 17, 1997 and is assigned ATCC deposit no. ATCC 209403.

Analysis of the amino acid sequence of the full-length PRO257polypeptide suggests that portions of it possess significant homology tothe ebnerin protein, thereby indicating that PRO257 may be a novelprotein member related to the ebnerin protein.

Example 6 Isolation of cDNA Clones Encoding Human PRO268 Polypeptides[UNQ235]

A consensus DNA sequence was assembled relative to other EST sequencesusing phrap as described in Example 1 above. This consensus sequence isherein designated DNA35698. Based on the DNA35698 consensus sequence,oligonucleotides were synthesized: 1) to identify by PCR a cDNA librarythat contained the sequence of interest, and 2) for use as probes toisolate a clone of the full-length coding sequence for PRO268.

Forward and reverse PCR primers were synthesized:

forward PCR primer 1 5′-TGAGGTGGGCAAGCGGCGAAATG-3′ (SEQ ID NO: 115)forward PCR primer 2 5′-TATGTGGATCAGGACGTGCC-3′ (SEQ ID NO: 116)forward PCR primer 3 5′-TGCAGGGTTCAGTCTAGATTG-3′ (SEQ ID NO: 117)reverse PCR primer 5′-TTGAAGGACAAAGGCAATCTGCCAC-3′ (SEQ ID NO: 118)Additionally, a synthetic oligonucleotide hybridization probe wasconstructed from the consensus DNA35698 sequence which had the followingnucleotide sequence

hybridization probe (SEQ ID NO: 119)5′-GGAGTCTTGCAGTTCCCCTGGCAGTCCTGGTGCTGTTGCTTTGG G-3′

In order to screen several libraries for a source of a full-lengthclone, DNA from the libraries was screened by PCR amplification with thePCR primer pair identified above. A positive library was then used toisolate clones encoding the PRO268 gene using the probe oligonucleotideand one of the PCR primers.

RNA for construction of the cDNA libraries was isolated from human fetallung tissue.

DNA sequencing of the clones isolated as described above gave thefull-length DNA sequence for PRO268 [herein designated as DNA39427-1179](SEQ ID NO:5) and the derived protein sequence for PRO268.

The entire nucleotide sequence of DNA39427-1179 is shown in FIG. 5 (SEQID NO:5). Clone DNA39427-1179 contains a single open reading frame withan apparent translational initiation site at nucleotide positions 13-15and ending at the stop codon at nucleotide positions 853-855 (FIG. 5).The predicted polypeptide precursor is 280 amino acids long (FIG. 6; SEQID NO:6). Clone DNA39427-1179 has been deposited with ATCC on Oct. 17,1997 and is assigned ATCC deposit no. ATCC 209395.

Analysis of the amino acid sequence of the full-length PRO268polypeptide suggests that it possess significant homology to proteindisulfide isomerase, thereby indicating that PRO268 may be a novelprotein disulfide isomerase.

Example 7 Isolation of cDNA Clones Encoding Human PRO290 Polypeptides[UNQ253]

An expressed sequence tag (EST) DNA database (LIFESEQ®, IncytePharmaceuticals, Palo Alto, Calif.) was searched and an EST wasidentified that had homology to beige and FAN. An oligonucleotide probebased upon the identified EST sequence was then synthesized and used toscreen human fetal kidney cDNA libraries in an attempt to identify afull-length cDNA clone. The oligonucleotide probe had the followingsequence:

(SEQ ID NO: 120) 5′ TGACTGCACTACCCCGTGGCAAGCTGTTGAGCCAGCTCAGCTG 3′.

RNA for construction of cDNA libraries was isolated from human fetalkidney tissue. The cDNA libraries used to isolate the cDNA clonesencoding human PRO290 were constructed by standard methods usingcommercially available reagents such as those from Invitrogen, SanDiego, Calif. The cDNA was primed with oligo dT containing a NotI site,linked with blunt to SalI hemikinased adaptors, cleaved with NotI, sizedappropriately by gel electrophoresis, and cloned in a definedorientation into a suitable cloning vector (such as pRKB or pRKD; pRK5Bis a precursor of pRK5D that does not contain the SfiI site; see, Holmeset al., Science 253:1278-1280 (1991)) in the unique XhoI and NotI.

A cDNA clone was identified and sequenced in entirety. The entirenucleotide sequence of DNA35680-1212 is shown in FIG. 7 (SEQ ID NO:7).Clone DNA35680-1212 contains a single open reading frame with anapparent translational initiation site at nucleotide positions 293-295,and a stop codon at nucleotide positions 3302-3304 (FIG. 7; SEQ IDNO:7). The predicted polypeptide precursor is 1003 amino acids long(FIG. 8; SEQ ID NO:8).

It is currently believed that the PRO290 polypeptide is related to FANand/or beige. Clone DNA35680-1212 has been deposited with ATCC on Apr.21, 1998 and is assigned ATCC deposit no. 209790. It is understood thatthe deposited clone has the actual correct sequence rather than therepresentations provided herein. The full-length PRO290 protein shown inFIG. 8 has an estimated molecular weight of about 112,013 daltons and apI of about 6.4.

Example 8 Isolation of cDNA Clones Encoding Human PRO363 Polypeptides[UNQ318]

A consensus sequence was obtained relative to a variety of EST sequencesas described in Example 1 above, wherein the consensus sequence obtainedis herein designated DNA42828. Based on the DNA42828 consensus sequence,oligonucleotides were synthesized: 1) to identify by PCR a cDNA librarythat contained the sequence of interest, and 2) for use as probes toisolate a clone of the full-length coding sequence for PRO363.

A pair of PCR primers (forward and reverse) were synthesized:

forward PCR primer (42828.f1) 5′-CCAGTGCACAGCAGGCAACGAAGC-3′(SEQ ID NO: 121) reverse PCR primer (42828.r1)5′-ACTAGGCTGTATGCCTGGGTGGGC-3′ (SEQ ID NO: 122)Additionally, a synthetic oligonucleotide hybridization probe wasconstructed from the consensus DNA42828 sequence which had the followingnucleotide sequence

hybridization probe (42828.p1) (SEQ ID NO: 123)5′-GTATGTACAAAGCATCGGCATGGTTGCAGGAGCAGTGACAGGC-3′

In order to screen several libraries for a source of a full-lengthclone, DNA from the libraries was screened by PCR amplification with thePCR primer pair identified above. A positive library was then used toisolate clones encoding the PRO363 gene using the probe oligonucleotideand one of the PCR primers. RNA for construction of the cDNA librarieswas isolated from human fetal kidney tissue (LIB227).

DNA sequencing of the clones isolated as described above gave thefull-length DNA sequence for PRO363 [herein designated as UNQ318(DNA45419-1252)] (SEQ ID NO:11) and the derived protein sequence forPRO363.

The entire nucleotide sequence of UNQ318 (DNA45419-1252) is shown inFIG. 11 (SEQ ID NO:11). Clone UNQ318 (DNA45419-1252) contains a singleopen reading frame with an apparent translational initiation site atnucleotide positions 190-192 and ending at the stop codon at nucleotidepositions 1309-1311 (FIG. 11). The predicted polypeptide precursor is373 amino acids long (FIG. 12). The full-length PRO363 protein shown inFIG. 12 has an estimated molecular weight of about 41,281 daltons and apI of about 8.33. A transmembrane domain exists at amino acids 221 to254 of the amino acid sequence shown in FIG. 12 (SEQ ID NO:12). ThePRO363 polypeptide also possesses at least two myelin P0 protein domainsfrom about amino acids 15 to 56 and from about amino acids 87 to 116.Clone UNQ318 (DNA45419-1252) has been deposited with ATCC on Feb. 5,1998 and is assigned ATCC deposit no. 209616.

Analysis of the amino acid sequence of the full-length PRO363polypeptide suggests that it possesses significant sequence similarityto the cell surface protein HCAR, thereby indicating that PRO363 may bea novel HCAR homolog. More specifically, an analysis of the Dayhoffdatabase (version 35.45 SwissProt 35) evidenced significant homologybetween the PRO363 amino acid sequence and the following Dayhoffsequences, HS46 KDA_(—)1, HSU90716_(—)1, MMCARH_(—)1, MMCARHOM_(—)1,MMU90715_(—)1, A33_HUMAN, P_W14146, P_W14158, A42632 and B42632.

Example 9 Isolation of cDNA Clones Encoding Human PRO365 Polypeptides[UNQ320]

A consensus DNA sequence was assembled relative to other EST sequencesusing phrap as described in Example 1 above. This consensus sequence isherein designated DNA35613. Based on the DNA35613 consensus sequence,oligonucleotides were synthesized: 1) to identify by PCR a cDNA librarythat contained the sequence of interest, and 2) for use as probes toisolate a clone of the full-length coding sequence for PRO365.

Forward and reverse PCR primers were synthesized as follows:

forward PCR primer 5′-AATGTGACCACTGGACTCCC-3′ (SEQ ID NO: 124)forward PCR primer 5′-AGGCTTGGAACTCCCTTC-3′ (SEQ ID NO: 125)reverse PCR primer 5′-AAGATTCTTGAGCGATTCCAGCTG-3′ (SEQ ID NO: 126)Additionally, a synthetic oligonucleotide hybridization probe wasconstructed from the consensus DNA35613 sequence which had the followingnucleotide sequence hybridization probe

hybridization probe (SEQ ID NO: 127)5′-AATCCCTGCTCTTCATGGTGACCTATGACGACGGAAGCACAAGACT G-3′

In order to screen several libraries for a source of a full-lengthclone, DNA from the libraries was screened by PCR amplification with oneof the PCR primer pairs identified above. A positive library was thenused to isolate clones encoding the PRO365 gene using the probeoligonucleotide and one of the PCR primers. RNA for construction of thecDNA libraries was isolated from human fetal kidney tissue.

DNA sequencing of the clones isolated as described above gave thefull-length DNA sequence for PRO365 [herein designated as DNA46777-1253](SEQ ID NO:13) and the derived protein sequence for PRO365.

The entire nucleotide sequence of DNA46777-1253 is shown in FIG. 13 (SEQID NO:13). Clone DNA46777-1253 contains a single open reading frame withan apparent translational initiation site at nucleotide positions 15-17and ending at the stop codon at nucleotide positions 720-722 (FIG. 13).The predicted polypeptide precursor is 235 amino acids long (FIG. 14;SEQ ID NO:14). Important regions of the polypeptide sequence encoded byclone DNA46777-1253 have been identified and include the following: asignal peptide corresponding to amino acids 1-20, the start of themature protein corresponding to amino acid 21, and multiple potentialN-glycosylation sites as shown in FIG. 14. Clone DNA46777-1253 has beendeposited with ATCC on Feb. 5, 1998 and is assigned ATCC deposit no.ATCC 209619.

Analysis of the amino acid sequence of the full-length PRO365polypeptide suggests that portions of it possess significant homology tothe human 2-19 protein, thereby indicating that PRO365 may be a novelhuman 2-19 protein homolog.

Example 10 Isolation of cDNA Clones Encoding Human PRO382 Polypeptides[UNQ323]

A consensus sequence was obtained relative to a variety of EST sequencesas described in Example 1 above, wherein the consensus sequence obtainedis herein designated DNA30892. Based on the DNA30892 consensus sequence,oligonucleotides were synthesized: 1) to identify by PCR a cDNA librarythat contained the sequence of interest, and 2) for use as probes toisolate a clone of the full-length coding sequence for PRO382.

A pair of PCR primers (forward and reverse) were synthesized:

forward PCR primer 5′-TGACATCGCCCTTATGAAGCTGGC-3′ (SEQ ID NO: 128)reverse PCR primer 5′-TACACGTCCCTGTGGTTGCAGATC-3′ (SEQ ID NO: 129)Additionally, a synthetic oligonucleotide hybridization probe wasconstructed from the consensus DNA30892 sequence which had the followingnucleotide sequence

hybridization probe (SEQ ID NO: 130)5′-CGTTCAATGCAGAAATGATCCAGCCTGTGTGCCTGCCCAACTCTGAA GAG-3′

In order to screen several libraries for a source of a full-lengthclone, DNA from the libraries was screened by PCR amplification with thePCR primer pair identified above. A positive library was then used toisolate clones encoding the PRO382 gene using the probe oligonucleotideand one of the PCR primers. RNA for construction of the cDNA librarieswas isolated from human fetal kidney tissue (LIB227).

DNA sequencing of the clones isolated as described above gave thefull-length DNA sequence for PRO382 [herein designated as UNQ323(DNA45234-1277)] (SEQ ID NO:15) and the derived protein sequence forPRO382.

The entire nucleotide sequence of UNQ323 (DNA45234-1277) is shown inFIG. 15 (SEQ ID NO:15). Clone UNQ323 (DNA45234-1277) contains a singleopen reading frame with an apparent translational initiation site atnucleotide positions 126-128 and ending at the stop codon at nucleotidepositions 1485-1487 (FIG. 15). The predicted polypeptide precursor is453 amino acids long (FIG. 16; SEQ ID NO:16). The full-length PRO382protein shown in FIG. 16 has an estimated molecular weight of about49,334 daltons and a pI of about 6.32. Analysis of the native PRO382amino acid sequence shown in FIG. 16 (SEQ ID NO:16) indicates thepresence of a putative transmembrane domain from about amino acid 240 toabout amino acid 284, a putative signal peptide at about amino acid 1 toabout amino acid 20, a putative apple domain at about amino acid 386 toabout amino acid 419, a putative Kringle domain at about amino acid 394to about amino acid 406 and a histidine-containing protease active siteat about amino acid 253 to about amino acid 258. Clone UNQ323(DNA45234-1277) has been deposited with ATCC on Mar. 5, 1998 and isassigned ATCC deposit no. 209654.

Analysis of the amino acid sequence of the full-length PRO382polypeptide suggests that it possess significant homology to serineprotease proteins, thereby indicating that PRO382 may be a novel serineprotease. Specifically, an analysis of the Dayhoff database (version35.45 SwissProt 35) evidenced significant homology between the PRO382amino acid sequence and the following Dayhoff sequences, HSU75329_(—)1,ENTK_MOUSE, HEPS_HUMAN, AF030065_(—)1, HEPS_RAT, PLMN_PIG, P_R89430,P_R89435, PLMN_HORSE, PLMN_BOVIN and P_R83959.

Example 11 Isolation of cDNA Clones Encoding Human PRO444 Polypeptides[UNQ328]

A cDNA sequence isolated in the amylase screen described in Example 2above was designated DNA13121. Oligonucleotide probes were generated tothis sequence and used to screen a human fetal lung library (LIB25)prepared as described in paragraph 1 of Example 2 above. The cloningvector was pRK5B (pRK5B is a precursor of pRK5D that does not containthe SfiI site; see, Holmes et al., Science, 253:1278-1280 (1991)), andthe cDNA size cut was less than 2800 bp.

A full length clone was identified that contained a single open readingframe with an apparent translational initiation site at nucleotidepositions 608-610 and ending at the stop codon found at nucleotidepositions 959-961 (FIG. 17, SEQ ID NO:17). The predicted polypeptideprecursor is 117 amino acids long, has a calculated molecular weight ofapproximately 12,692 daltons and an estimated pI of approximately 7.50.Analysis of the full-length PRO444 sequence shown in FIG. 18 (SEQ IDNO:18) evidences the presence of a signal peptide at amino acid 1 toabout amino acid 16. An analysis of the Dayhoff database (version 35.45SwissProt 35) evidenced homology between the PRO444 amino acid sequenceand the following Dayhoff sequences: CEF44D12_(—)8, P_R88452,YNE1_CAEEL, A47312, AF009957_(—)1, and A06133_(—)1.

Clone DNA26846-1397 was deposited with the ATCC on Oct. 27, 1998 and isassigned ATCC deposit no. 203406.

Example 12 Isolation of cDNA Clones Encoding Human PRO705 Polypeptides[UNQ369]

A consensus sequence was obtained relative to a variety of EST sequencesas described in Example 1 above, wherein the consensus sequence obtainedis herein designated DNA43437. Based on the DNA43437 consensus sequence,oligonucleotides were synthesized: 1) to identify by PCR a cDNA librarythat contained the sequence of interest, and 2) for use as probes toisolate a clone of the full-length coding sequence for PRO705.

A pair of PCR primers (forward and reverse) were synthesized:

forward PCR primer 5′-AAGCGTGACAGCGGGCACGTC-3′ (SEQ ID NO: 131)reverse PCR primer 5′-TGCACAGTCTCTGCAGTGCCCAGG-3′ (SEQ ID NO: 132)Additionally, a synthetic oligonucleotide hybridization probe wasconstructed from the consensus DNA43437 sequence which had the followingnucleotide sequence

hybridization probe (43437.p1) (SEQ ID NO: 133)5′-GAATGCTGGAACGGGCACAGCAAAGCCAGATACTTGCCTG-3′

In order to screen several libraries for a source of a full-lengthclone, DNA from the libraries was screened by PCR amplification with thePCR primer pair identified above. A positive library was then used toisolate clones encoding the PRO705 gene using the probe oligonucleotideand one of the PCR primers. RNA for construction of the cDNA librarieswas isolated from human fetal kidney tissue (LIB227).

DNA sequencing of the clones isolated as described above gave thefull-length DNA sequence for PRO705 [herein designated as UNQ369(DNA50914-1289)] (SEQ ID NO:19) and the derived protein sequence forPRO705.

The entire nucleotide sequence of UNQ369 (DNA50914-1289) is shown inFIG. 19 (SEQ ID NO:19). Clone UNQ369 (DNA50914-1289) contains a singleopen reading frame with an apparent translational initiation site atnucleotide positions 566-568 and ending at the stop codon at nucleotidepositions 2231-2233 (FIG. 19). The predicted polypeptide precursor is555 amino acids long (FIG. 20; SEQ ID NO:20). The full-length PRO705protein shown in FIG. 20 has an estimated molecular weight of about62,736 daltons and a pI of about 5.36. Analysis of the full-lengthPRO705 sequence as shown in FIG. 20 evidences the presence of thefollowing: a signal peptide from about amino acid 1 to about amino acid23, a eukaryotic DNA topoisomerase I active site from about amino acid418 to about amino acid 436, and various regions that show homology tovarious glypican proteins from about amino acid 237 to about amino acid279, about amino acid 421 to about amino acid 458, about amino acid 53to about amino acid 74, about amino acid 466 to about amino acid 504,about amino acid 308 to about amino acid 355, about amino acid 104 toabout amino acid 156 and about amino acid 379 to about amino acid 410.Clone UNQ369 (DNA50914-1289) has been deposited with ATCC on Mar. 31,1998 and is assigned ATCC deposit no. 209722.

Analysis of the amino acid sequence of the full-length PRO705polypeptide suggests that it possesses significant sequence similarityto the K-glypican protein, thereby indicating that PRO705 may be a novelglypican protein family member. More specifically, an analysis of theDayhoff database (version 35.45 SwissProt 35) evidenced significanthomology between the PRO705 amino acid sequence and the followingDayhoff sequences, GPCK_MOUSE, GLYP_CHICK, GLYP_RAT, GLYP_HUMAN,GPC2_RAT, GPC5_HUMAN, GPC3_HUMAN, GPC3_RAT, P_R30168 and CEC03H12_(—)2.

Example 13 Isolation of cDNA Clones Encoding Human PRO1071 Polypeptides[UNQ528]

A consensus sequence was obtained relative to a variety of EST sequencesas described in Example 1 above, wherein the consensus sequence obtainedis herein designated DNA53035. Based on the DNA53035 consensus sequence,it was determined that that consensus sequence shared significantsequence identity with Incyte EST clone no. 2872569, a clone that uponreview appeared to encode a full length protein. As such, Incyte ESTclone no. 2872569 was purchased and its insert was obtained andsequenced so as to confirm the proper sequence. This sequence is hereindesignated UNQ528 or DNA58847-1383.

DNA sequencing of the clone isolated as described above gave thefull-length DNA sequence for PRO1071 [herein designated as UNQ528(DNA58847-1383)] (SEQ ID NO:21) and the derived protein sequence forPRO1071.

The entire nucleotide sequence of UNQ528 (DNA58847-1383) is shown inFIG. 21 (SEQ ID NO:21). Clone UNQ528 (DNA58848-1383) contains a singleopen reading frame with an apparent translational initiation site atnucleotide positions 133-135 and ending at the stop codon at nucleotidepositions 1708-1710 (FIG. 21). The predicted polypeptide precursor is525 amino acids long (FIG. 22; SEQ ID NO:22). The full-length PRO1071protein shown in FIG. 22 has an estimated molecular weight of about58,416 daltons and a pI of about 6.62. Analysis of the full-lengthPRO1071 sequence shown in FIG. 22 (SEQ ID NO:22) evidences the presenceof the following: a signal peptide from about amino acid 1 to aboutamino acid 25, a potential N-glycosylation site from about amino acid251 to about amino acid 254, a thrombospondin-1 homology block fromabout amino acid 385 to about amino acid 399 and von Willibrands factortype C homology blocks from about amino acid 385 to about amino acid399, from about amino acid 445 to about amino acid 459 and from aboutamino acid 42 to about amino acid 56. Clone UNQ528 (DNA58847-1383) hasbeen deposited with ATCC on May 20, 1998 and is assigned ATCC depositno. 209879.

Analysis of the amino acid sequence of the full-length PRO1071polypeptide suggests that it possesses significant sequence similarityto the thrombospondin protein, thereby indicating that PRO1071 may be anovel thrombospondin homolog. More specifically, an analysis of theDayhoff database (version 35.45 SwissProt 35) evidenced significanthomology between the PRO1071 amino acid sequence and the followingDayhoff sequences, AB002364_(—)1, D67076_(—)1, BTPCINPGN_(—)1,CET13H10_(—)1, CEF25H8_(—)5, CEF53B6_(—)2, CEC26C6_(—)6, HSSEMG_(—)1,CET21B6_(—)4 and BTY08561_(—)1.

Example 14 Isolation of cDNA Clones Encoding Human PRO1125 Polypeptides[UNQ563]

Use of the signal sequence algorithm described in Example 3 aboveallowed identification of a single EST cluster sequence from the Incytedatabase. This EST cluster sequence was then compared to a variety ofexpressed sequence tag (EST) databases which included public ESTdatabases (e.g., GenBank) and a proprietary EST DNA database (LIFESEQ®,Incyte Pharmaceuticals, Palo Alto, Calif.) to identify existinghomologies. The homology search was performed using the computer programBLAST or BLAST2 (Altshul et al., Methods in Enzymology 266:460-480(1996)). Those comparisons resulting in a BLAST score of 70 (or in somecases 90) or greater that did not encode known proteins were clusteredand assembled into a consensus DNA sequence with the program “phrap”(Phil Green, University of Washington, Seattle, Wash.). The consensussequence obtained therefrom is herein designated DNA56540.

In light of an observed sequence homology between the DNA56540 consensussequence and an EST sequence encompassed within the Incyte EST clone no.1486114, the Incyte EST clone 1486114 was purchased and the cDNA insertwas obtained and sequenced. It was found that this insert encoded afull-length protein. The sequence of this cDNA insert is shown in FIG.23 and is herein designated as DNA60615-1482.

The full length clone shown in FIG. 23 contained a single open readingframe with an apparent translational initiation site at nucleotidepositions 47-49 and ending at the stop codon found at nucleotidepositions 1388-1390 (FIG. 23; SEQ ID NO:23). The predicted polypeptideprecursor (FIG. 24, SEQ ID NO:24) is 447 amino acids long. PRO1125 has acalculated molecular weight of approximately 49,798 daltons and anestimated pI of approximately 9.78. Clone DNA60619-1482 has beendeposited with ATCC on Jun. 16, 1998 and is assigned ATCC deposit no.209993. It is understood that the clone has the actual sequence and thatthe sequences herein are representations based on current techniqueswhich may be prone to minor errors.

Based on a WU-BLAST2 sequence alignment analysis (using the ALIGNcomputer program) of the full-length sequence, PRO1125 shows somesequence identity with the following Dayhoff designations: RCO1NEUCR;S58306; PKWA_THECU; S76086; P_R85881; HET1_PODAN; SPU92792_(—)1;APAF_HUMAN; S76414 and S59317.

Example 15 Isolation of cDNA Clones Encoding Human PRO1134 Polypeptides[UNQ572]

Use of the signal sequence algorithm described in Example 3 aboveallowed identification of an EST cluster sequence from the Incytedatabase, designated 7511. This EST cluster sequence was then comparedto a variety of expressed sequence tag (EST) databases which includedpublic EST databases (e.g., GenBank) and a proprietary EST DNA database(Lifeseq®, Incyte Pharmaceuticals, Palo Alto, Calif.) to identifyexisting homologies. The homology search was performed using thecomputer program BLAST or BLAST2 (Altshul et al., Methods in Enzymology266:460-480 (1996)). Those comparisons resulting in a BLAST score of 70(or in some cases 90) or greater that did not encode known proteins wereclustered and assembled into a consensus DNA sequence with the program“phrap” (Phil Green, University of Washington, Seattle, Wash.). Theconsensus sequence obtained therefrom is herein designated DNA55725. Twoproprietary Genentech EST sequences were employed in the assembly.

In light of an observed sequence homology between the DNA55725 consensussequence and an EST sequence encompassed within the Merck EST clone no.H94897, the Merck EST clone H94897 was purchased and the cDNA insert wasobtained and sequenced. It was found that this insert encoded afull-length protein. The sequence of this cDNA insert is shown in FIG.25 and is herein designated as DNA56865-1491.

Clone DNA56865-1491 contains a single open reading frame with anapparent translational initiation site at nucleotide positions 153-155and ending at the stop codon at nucleotide positions 1266-1268 (FIG. 25;SEQ ID NO:25). The predicted polypeptide precursor is 371 amino acidslong (FIG. 26; SEQ ID NO:26). The full-length PRO1134 protein shown inFIG. 26 has an estimated molecular weight of about 41,935 daltons and apI of about 9.58. Analysis of the full-length PRO1134 sequence shown inFIG. 26 (SEQ ID NO:26) evidences the presence of the following: a signalpeptide from about amino acid 1 to about amino acid 23, potentialN-glycosylation sites from about amino acid 103 to about amino acid 106,from about amino acid 249 to about amino acid 252 and from about aminoacid 257 to about amino acid 260, and an amino acid block havinghomology to tyrosinase CuA-binding region proteins from about amino acid280 to about amino acid 306. Clone DNA56865-1491 has been deposited withATCC on Jun. 23, 1998 and is assigned ATCC deposit no. 203022.

An analysis of the Dayhoff database (version 35.45 SwissProt 35), usinga WU-BLAST-2 sequence alignment analysis of the full-length sequenceshown in FIG. 26 (SEQ ID NO:26), evidenced significant homology betweenthe PRO1134 amino acid sequence and the following Dayhoff sequences:F20P5_(—)18, AC002396_(—)10, S47847, C64146, GSPA_BACSU, P_W10564,RFAI_ECOL1, Y258_HAEIN, RFAJ_SALTY and P_R32985.

Example 16 Isolation of cDNA Clones Encoding Human PRO1155 Polypeptides[UNQ585]

Use of the signal sequence algorithm described in Example 3 aboveallowed identification of a single EST cluster sequence from the Incytedatabase. This EST cluster sequence was then compared to a variety ofexpressed sequence tag (EST) databases which included public ESTdatabases (e.g., GenBank) and a proprietary EST DNA database (LIFESEQ®,Incyte Pharmaceuticals, Palo Alto, Calif.) to identify existinghomologies. The homology search was performed using the computer programBLAST or BLAST2 (Altshul et al., Methods in Enzymology 266:460-480(1996)). Those comparisons resulting in a BLAST score of 70 (or in somecases 90) or greater that did not encode known proteins were clusteredand assembled into a consensus DNA sequence with the program “phrap”(Phil Green, University of Washington, Seattle, Wash.). The consensussequence obtained therefrom is herein designated DNA56102.

In light of an observed sequence homology between the DNA56102 consensussequence and an EST sequence encompassed within the Incyte EST clone no.2858870, the Incyte EST clone 2858870 was purchased and the cDNA insertwas obtained and sequenced. It was found that this insert encoded afull-length protein. The sequence of this cDNA insert is shown in FIG.27 and is herein designated as DNA59849-1504.

The full length clone shown in FIG. 27 contained a single open readingframe with an apparent translational initiation site at nucleotidepositions 158-160 and ending at the stop codon found at nucleotidepositions 563-565 (FIG. 27; SEQ ID NO:27). The predicted polypeptideprecursor (FIG. 28, SEQ ID NO:28) is 135 amino acids long. PRO1155 has acalculated molecular weight of approximately 14,833 daltons and anestimated pI of approximately 9.78. Clone DNA59849-1504 has beendeposited with ATCC on Jun. 16, 1998 and is assigned ATCC deposit no.209986. It is understood that the actual clone has the correct sequencewhereas herein are only representations which are prone to minorsequencing errors.

Based on a WU-BLAST2 sequence alignment analysis (using the ALIGNcomputer program) of the full-length sequence, PRO1155 shows some aminoacid sequence identity with the following Dayhoff designations:TKNK_BOVIN; PVB19X587_(—)1; AF019049_(—)1; P_WO0948; S72864; P_WO0949;I62742; AF038501_(—)1; TKNG_HUMAN; and YAT1_RHOBL. Based on theinformation provided herein, PRO1155 may play a role in providingneuroprotection and cognitive enhancement.

Example 17 Isolation of cDNA Clones Encoding Human PRO1281 Polypeptides[UNQ651]

A consensus DNA sequence was assembled relative to other EST sequencesusing phrap as described in Example 1 above. This consensus sequence isdesignated herein as DNA35720. Based on the DNA35720 sequence,oligonucleotides were synthesized: 1) to identify by PCR a cDNA librarythat contained the sequence of interest, and 2) for use as probes toisolate a clone of the full-length coding sequence for PRO1281.

PCR primers (forward and reverse) were synthesized:

Forward PCR Primers:

5′-TGGAAGGCTGCCGCAACGACAATC-3′; (SEQ ID NO: 134)5′-CTGATGTGGCCGATGTTCTG-3′; (SEQ ID NO: 135) and5′-ATGGCTCAGTGTGCAGACAG-3′. (SEQ ID NO: 136)Reverse PCR Primers:

5′-GCATGCTGCTCCGTGAAGTAGTCC-3′; (SEQ ID NO: 137) and5′-ATGCATGGGAAAGAAGGCCTGCCC-3′. (SEQ ID NO: 138)Additionally, a synthetic oligonucleotide hybridization probe wasconstructed from the DNA35720 sequence which had the followingnucleotide sequence:

hybridization probe: (SEQ ID NO: 139)5′-TGCACTGGTGACCACGAGGGGGTGCACTATAGCCATCTGGAGCTGA G-3′.

In order to screen several libraries for a source of a full-lengthclone, DNA from the libraries was screened by PCR amplification with thePCR primer pairs identified above. A positive library was then used toisolate clones encoding the PRO1281 gene using the probe oligonucleotideand one of the PCR primers. RNA for construction of the cDNA librarieswas isolated human fetal liver.

DNA sequencing of the clones isolated as described above gave thefull-length DNA sequence for PRO1281 (designated herein as DNA59820-1549[FIG. 29, SEQ ID NO:29]; and the derived protein sequence for PRO1281.

The entire coding sequence of PRO1281 is shown in FIG. 29 (SEQ IDNO:29). Clone DNA59820-1549 contains a single open reading frame with anapparent translational initiation site at nucleotide positions 228-230and an apparent stop codon at nucleotide positions 2553-2555. Thepredicted polypeptide precursor is 775 amino acids long. The full-lengthPRO1281 protein shown in FIG. 30 has an estimated molecular weight ofabout 85,481 daltons and a pI of about 6.92. Additional features includea signal peptide at about amino acids 1-15; and potentialN-glycosylation sites at about amino acids 138-141 and 361-364.

An analysis of the Dayhoff database (version 35.45 SwissProt 35), usinga WU-BLAST2 sequence alignment analysis of the full-length sequenceshown in FIG. 30 (SEQ ID NO:30), revealed some sequence identity betweenthe PRO1281 amino acid sequence and the following Dayhoff sequences:S44860, CET24D1_(—)1, CEC38H2_(—)3, CAC2_HAECO, B3A2_HUMAN, S22373,CEF38A3_(—)2, CEC34F6_(—)2, CEC34F6_(—)3, and CELT22B11_(—)3.

Clone DNA59820-1549 has been deposited with ATCC on Aug. 18, 1998 and isassigned ATCC deposit no. 203129.

Example 18 Isolation of cDNA Clones Encoding Human PRO1343 Polypeptides[UNQ698]

A cDNA sequence isolated in the amylase screen described in Example 2above was found, by the WU-BLAST2 sequence alignment computer program,to have no significant sequence identity to any known human encodingnucleic acid. This cDNA sequence is herein designated DNA48921. Probeswere generated from the sequence of the DNA48921 molecule and used toscreen a human smooth muscle cell tissue library prepared as describedin paragraph 1 above. The cloning vector was pRK5B (pRK5B is a precursorof pRK5D that does not contain the SfiI site; see, Holmes et al.,Science, 253:1278-1280 (1991)), and the cDNA size cut was less than 2800bp.

The oligonucleotide probes employed were as follows:

forward PCR primer (48921.f1) (SEQ ID NO: 140)5′-CAATATGCATCTTGCACGTCTGG-3′ reverse PCR primer (48921.r1)(SEQ ID NO: 141) 5′-AAGCTTCTCTGCTTCCTTTCCTGC-3′hybridization probe (48921.p1) (SEQ ID NO: 142)5′-TGACCCCATTGAGAAGGTCATTGAAGGGATCAACCGAGGGCTG-3′

A full length clone was identified that contained a single open readingframe with an apparent translational initiation site at nucleotidepositions 71-73 and a stop signal at nucleotide positions 812-814 (FIG.31, SEQ ID NO:31). The predicted polypeptide precursor is 247 aminoacids long, has a calculated molecular weight of approximately 25,335daltons and an estimated pI of approximately 7.0. Analysis of thefull-length PRO1343 sequence shown in FIG. 32 (SEQ ID NO:32) evidencesthe presence of the following: a signal peptide from about amino acid 1to about amino acid 25 and a homologous region to circumsporozoiterepeats from about amino acid 35 to about amino acid 225. CloneDNA66675-1587 has been deposited with ATCC on Sep. 22, 1998 and isassigned ATCC deposit no. 203282.

An analysis of the Dayhoff database (version 35.45 SwissProt 35), usinga WU-BLAST2 sequence alignment analysis of the full-length sequenceshown in FIG. 32 (SEQ ID NO:32), evidenced significant homology betweenthe PRO1343 amino acid sequence and the following Dayhoff sequences:CSP_PLACC, CEF25H8_(—)2, U88974_(—)40, BNAMRNAA_(—)1, BOBOPC3_(—)1,S58135, AF061832_(—)1, BHU52040_(—)1, HUMPROFILE_(—)1 and MTV023_(—)14.

Additionally, an Incyte EST clone (Incyte EST clone no. 4701148) havinghomology to the DNA48921 sequence was obtained and the insert sequenced,thereby giving rise to the DNA66675-1587 sequence shown in FIG. 31.

Example 19 Isolation of cDNA Clones Encoding Human PRO1379 Polypeptides[UNQ716]

A consensus DNA sequence was assembled relative to other EST sequencesusing phrap as described in Example 1 above. This consensus sequence isdesignated herein DNA45232. Based on the DNA45232 consensus sequence,oligonucleotides were synthesized: 1) to identify by PCR a cDNA librarythat contained the sequence of interest, and 2) for use as probes toisolate a clone of the full-length coding sequence for PRO1379.

PCR primers (forward and reverse) were synthesized:

forward PCR primer 5′-TGGACACCGTACCCTGGTATCTGC-3′ (SEQ ID NO: 143)reverse PCR primer 5′-CCAACTCTGAGGAGAGCAAGTGGC-3′ (SEQ ID NO: 144)

Additionally, a synthetic oligonucleotide hybridization probe wasconstructed from the consensus DNA45232 sequence which had the followingnucleotide sequence:

hybridization probe (SEQ ID NO: 145)5′-TGTATGTGCACACCCTCACCATCACCTCCAAGGGCAAGGAGAA C-3′.

In order to screen several libraries for a source of a full-lengthclone, DNA from the libraries was screened by PCR amplification with thePCR primer pair identified above. A positive library was then used toisolate clones encoding the PRO1379 gene using the probe oligonucleotideand one of the PCR primers. RNA for construction of the cDNA librarieswas isolated human fetal kidney tissue.

DNA sequencing of the clones isolated as described above gave thefull-length DNA sequence for PRO1379 which is designated herein asDNA59828-1608 and shown in FIG. 33 (SEQ ID NO:33); and the derivedprotein sequence for PRO1379 (SEQ ID NO:34).

The entire coding sequence of PRO1379 is shown in FIG. 33 (SEQ IDNO:33). Clone DNA59828-1608 contains a single open reading frame with anapparent translational initiation site at nucleotide positions 10-12 andan apparent stop codon at nucleotide positions 1732-1734. The predictedpolypeptide precursor is 574 amino acids long. The full-length PRO1379protein shown in FIG. 34 has an estimated molecular weight of about65,355 daltons and a pI of about 8.73. Additional features include asignal peptide at about amino acids 1-17 and potential N-glycosylationsites at about amino acids 160-163, 287-290, and 323-326.

An analysis of the Dayhoff database (version 35.45 SwissProt 35), usinga WU-BLAST2 sequence alignment analysis of the full-length sequenceshown in FIG. 34 (SEQ ID NO:34), revealed some homology between thePRO1379 amino acid sequence and the following Dayhoff sequences:YHY8_YEAST, AF040625_(—)1, HP714394_(—)1, and HIV18U45630_(—)1.

Clone DNA59828-1608 has been deposited with ATCC on Aug. 25, 1998 and isassigned ATCC deposit no. 203158.

Example 20 Isolation of cDNA Clones Encoding Human PRO1380 Polypeptides[UNQ717]

A cDNA sequence isolated in the amylase screen described in Example 2above is herein designated DNA45776. Based on the DNA45776 sequence,oligonucleotide probes were generated and used to screen a human retinalibrary prepared as described in paragraph 1 of Example 2 above. Thecloning vector was pRK5B (pRK5B is a precursor of pRK5D that does notcontain the SfiI site; see, Holmes et al., Science, 253:1278-1280(1991)), and the cDNA size cut was less than 2800 bp.

PCR primers (forward and reverse) were synthesized:

forward PCR primer (45776.f1) 5′-TTTTGCGGTCACCATTGTCTGC-3′(SEQ ID NO: 146) and reverse PCR primer (45776.r1)5′-CGTAGGTGACACAGAAGCCCAGG-3′. (SEQ ID NO: 147)Additionally, a synthetic oligonucleotide hybridization probe wasconstructed from the DNA45776 sequence which had the followingnucleotide sequence:

hybridization probe (45776.p1) (SEQ ID NO: 148)5′-TACGGCATGACCGGCTCCTTTCCTATGAGGAACTCCCAGGCACTGAT AT-3′.

In order to screen several libraries for a source of a full-lengthclone, DNA from the libraries was screened by PCR amplification with thePCR primer pair identified above. A positive library was then used toisolate clones encoding the PRO1380 gene using the probe oligonucleotideand one of the PCR primers.

A full length clone was identified that contained a single open readingframe with an apparent translational initiation site at nucleotidepositions 36-38, and a stop signal at nucleotide positions 1461-1463(FIG. 35; SEQ ID NO:35). The predicted polypeptide precursor is 470amino acids long has a calculated molecular weight of approximately51,715 daltons and an estimated pI of approximately 7.86. Additionalfeatures include transmembrane domains at about amino acids 50-74,105-127, 135-153, 163-183, 228-252, 305-330, and 448-472; potentialN-glycosylation sites at about amino acids 14-17 and 84-87; and adihydrofolate reductase signature at about amino acids 60-68.

An analysis of the Dayhoff database (version 35.45 SwissProt 35), usinga WU-BLAST2 sequence alignment analysis of the full-length sequenceshown in FIG. 36 (SEQ ID NO:36), evidenced homology between the PRO1380amino acid sequence and the following Dayhoff sequences: HSU81375_(—)1,CEZK809_(—)6, CEK02E11_(—)1, AF034102_(—)1, JC4196, CEF36H2_(—)2,P_R92315, YAC2_YEAST, F1707_(—)13, and CEF44D12_(—)3.

Clone DNA60740-1615 was deposited with the ATCC on Nov. 3, 1998, and isassigned ATCC deposit no. 203456.

Example 21 Isolation of cDNA Clones Encoding Human PRO1387 Polypeptides[UNQ722]

Use of the signal sequence algorithm described in Example 3 aboveallowed identification of a single EST cluster sequence from the Incytedatabase. This EST cluster sequence was then compared to a variety ofexpressed sequence tag (EST) databases which included public ESTdatabases (e.g., GenBank) and a proprietary EST DNA database (LIFESEQ®,Incyte Pharmaceuticals, Palo Alto, Calif.) to identify existinghomologies. The homology search was performed using the computer programBLAST or BLAST2 (Altshul et al., Methods in Enzymology 266:460-480(1996)). Those comparisons resulting in a BLAST score of 70 (or in somecases 90) or greater that did not encode known proteins were clusteredand assembled into a consensus DNA sequence with the program “phrap”(Phil Green, University of Washington, Seattle, Wash.). The consensussequence obtained therefrom is herein designated DNA56259.

In light of an observed sequence homology between the DNA56259 consensussequence and an EST sequence encompassed within the Incyte EST clone no.3507924, the Incyte EST clone 3507924 was purchased and the cDNA insertwas obtained and sequenced. It was found that this insert encoded afull-length protein. The sequence of this cDNA insert is shown in FIG.37 and is herein designated as DNA68872-1620.

Clone DNA68872-1620 contains a single open reading frame with anapparent translational initiation site at nucleotide positions 85-87 andending at the stop codon at nucleotide positions 1267-1269 (FIG. 37; SEQID NO:37). The predicted polypeptide precursor is 394 amino acids long(FIG. 38). The full-length PRO1387 protein shown in FIG. 38 has anestimated molecular weight of about 44,339 daltons and a pI of about7.10. Analysis of the full-length PRO1387 sequence shown in FIG. 38 (SEQID NO:38) evidences the presence of the following: a signal peptide fromabout amino acid 1 to about amino acid 19, a transmembrane domain fromabout amino acid 275 to about amino acid 296, potential N-glycosylationsites from about amino acid 76 to about amino acid 79, from about aminoacid 231 to about amino acid 234, from about amino acid 302 to aboutamino acid 305, from about amino acid 307 to about amino acid 310 andfrom about amino acid 376 to about amino acid 379, and amino acidsequence blocks having homology to myelin p0 protein from about aminoacid 210 to about amino acid 239 and from about amino acid 92 to aboutamino acid 121. Clone DNA68872-1620 has been deposited with ATCC on Aug.25, 1998 and is assigned ATCC deposit no. 203160.

An analysis of the Dayhoff database (version 35.45 SwissProt 35), usinga WU-BLAST2 sequence alignment analysis of the full-length sequenceshown in FIG. 38 (SEQ ID NO:38), evidenced significant homology betweenthe PRO1387 amino acid sequence and the following Dayhoff sequences:P_W36955, MYP0_HETFR, HS46 KDA_(—)1, AF049498_(—)1, MYO0_HUMAN,AF030454_(—)1, A53268, SHPTCRA_(—)1, P_W14146 and GEN12838.

Example 22 Isolation of cDNA Clones Encoding Human PRO1419 Polypeptides[UNQ733]

Use of the signal sequence algorithm described in Example 3 aboveallowed identification of an EST cluster sequence from the Incytedatabase. This EST cluster sequence was then compared to a variety ofexpressed sequence tag (EST) databases which included public ESTdatabases (e.g., GenBank) and a proprietary EST DNA database (LIFESEQ®,Incyte Pharmaceuticals, Palo Alto, Calif.) to identify existinghomologies. One or more of the ESTs was derived from a diseased tonsiltissue library. The homology search was performed using the computerprogram BLAST or BLAST2 (Altshul et al., Methods in Enzymology266:460-480 (1996)). Those comparisons resulting in a BLAST score of 70(or in some cases 90) or greater that did not encode known proteins wereclustered and assembled into a consensus DNA sequence with the program“phrap” (Phil Green, University of Washington, Seattle, Wash.). Theconsensus sequence obtained therefrom is herein designated DNA59761.

In light of an observed sequence homology between the DNA59761 sequenceand an EST sequence contained within the Incyte EST 3815008, the cloneincluding this EST was purchased and the cDNA insert was obtained andsequenced. The sequence of this cDNA insert is shown in FIG. 39 and isherein designated as DNA71290-1630.

The full length clone shown in FIG. 39 contained a single open readingframe with an apparent translational initiation site at nucleotidepositions 86-88 and ending at the stop codon found at nucleotidepositions 341-343 (FIG. 39; SEQ ID NO:39). The predicted polypeptideprecursor (FIG. 40, SEQ ID NO:40) is 85 amino acids long with the signalpeptide at about amino acids 1-17 of SEQ ID NO:40. PRO1419 has acalculated molecular weight of approximately 9,700 daltons and anestimated pI of approximately 9.55. Clone DNA71290-1630 was depositedwith the ATCC on Sep. 22, 1998 and is assigned ATCC deposit no. 203275.

An analysis of the Dayhoff database (version 35.45 SwissProt 35), usinga WU-BLAST2 sequence alignment analysis of the full-length sequenceshown in FIG. 40 (SEQ ID NO:40), revealed sequence identity between thePRO1419 amino acid sequence and the following Dayhoff sequences (dataincorporated herein): S07975 (B3-hordein), C48232, HOR7_HORVU, GEN11764,S14970, AF020312_(—)1, STAJ3220_(—)1, CER07E3_(—)1, CEY37A1B_(—)4, andATAC00423810.

Example 23 Isolation of cDNA Clones Encoding Human PRO1433 Polypeptides[UNQ738]

A consensus DNA sequence was assembled relative to other EST sequencesusing phrap as described in Example 1 above. This consensus sequence isherein designated DNA45230. Based on the DNA45230 consensus sequence,oligonucleotides were synthesized: 1) to identify by PCR a cDNA librarythat contained the sequence of interest, and 2) for use as probes toisolate a clone of the full-length coding sequence for PRO1433.

PCR primers (forward and reverse) were synthesized:

forward PCR primer (45230.f1) 5′-GCTGACCTGGTTCCCATCTACTCC-3′(SEQ ID NO: 149) reverse PCR primer (45230.r1)5′-CCCACAGACACCCATGACACTTCC-3′ (SEQ ID NO: 150)Additionally, a synthetic oligonucleotide hybridization probe wasconstructed from the consensus DNA45230 sequence which had the followingnucleotide sequence

hybridization probe (45230.p1) (SEQ ID NO: 151)5′-AAGAATGAATTGTACAAAGCAGGTGATCTTCGAGGAGGGCTCCTGGG GCC-3′

In order to screen several libraries for a source of a full-lengthclone, DNA from the libraries was screened by PCR amplification with thePCR primer pair identified above. A positive library was then used toisolate clones encoding the PRO1433 gene using the probe oligonucleotideand one of the PCR primers. RNA for construction of the cDNA librarieswas isolated from human adrenal gland tissue.

DNA sequencing of the clones isolated as described above gave thefull-length DNA sequence for PRO1433 (designated herein as DNA71184-1634[FIG. 41, SEQ ID NO:41]; and the derived protein sequence for PRO1433.

The entire nucleotide sequence of DNA71184-1634 is shown in FIG. 41 (SEQID NO:41). Clone DNA71184-1634 contains a single open reading frame withan apparent translational initiation site at nucleotide positions185-187 and ending at the stop codon at nucleotide positions 1349-1351(FIG. 41). The predicted polypeptide precursor is 388 amino acids long(FIG. 42). The full-length PRO1433 protein shown in FIG. 42 has anestimated molecular weight of about 43,831 daltons and a pI of about9.64. Analysis of the full-length PRO1433 sequence shown in FIG. 42 (SEQID NO:42) evidences the presence of the following: a transmembranedomain from about amino acid 76 to about amino acid 97, potentialN-glycosylation sites from about amino acid 60 to about amino acid 63,from about amino acid 173 to about amino acid 176 and from about aminoacid 228 to about amino acid 231 and potential N-myristolation sitesfrom about amino acid 10 to about amino acid 15, from about amino acid41 to about amino acid 46, from about amino acid 84 to about amino acid89, from about amino acid 120 to about amino acid 125, from about aminoacid 169 to about amino acid 174, from about amino acid 229 to aboutamino acid 234, from about amino acid 240 to about amino acid 245, fromabout amino acid 318 to about amino acid 323 and from about amino acid378 to about amino acid 383. Clone DNA71184-1634 has been deposited withATCC on Sep. 22, 1998 and is assigned ATCC deposit no. 203266.

An analysis of the Dayhoff database (version 35.45 SwissProt 35), usinga WU-BLAST2 sequence alignment analysis of the full-length sequenceshown in FIG. 42 (SEQ ID NO:42), evidenced significant homology betweenthe PRO1433 amino acid sequence and the following Dayhoff sequences:CELW01A11_(—)4, CEF59A1_(—)4, S67138, MTV050_(—)3, S75135 and S12411.

Example 24 Isolation of cDNA Clones Encoding Human PRO1474 Polypeptides[UNQ745]

An expressed sequence tag (EST) DNA database (LIFESEQ®, IncytePharmaceuticals, Palo Alto, Calif.) was searched and an EST wasidentified. This EST showed homology to pancreatic secretory trypsininhibitor.

The clone which included this EST was purchased from Incyte (it camefrom a uterine cervical tissue library) and sequenced in full to revealthe nucleic acid of SEQ ID NO:43, which encodes PRO1474.

The entire nucleotide sequence of PRO1474 is shown in FIG. 43 (SEQ IDNO:43). Clone DNA73739-1645 contains a single open reading frame with anapparent translational initiation site at nucleotide positions 45-47 anda stop codon at nucleotide positions 300-302 (FIG. 43; SEQ ID NO:43).The predicted polypeptide precursor is 85 amino acids long. As indicatedin FIG. 44, the Kazal serine protease inhibitor family signature beginsat about amino acid 45 of SEQ ID NO:44. Also indicated in FIG. 44 is aregion conserved in integrin alpha chains (beginning at about amino acid32 of SEQ ID NO:44). Clone DNA73739-1645 has been deposited with theATCC on Sep. 22, 1998 and is assigned ATCC deposit no. 203270. Thefull-length PRO1474 protein shown in FIG. 44 has an estimated molecularweight of about 9,232 daltons and a pI of about 7.94.

An analysis of the Dayhoff database (version 35.45 SwissProt 35), usinga WU-BLAST2 sequence alignment analysis of the full-length sequenceshown in FIG. 44 (SEQ ID NO:44), revealed sequence identity between thePRO1474 amino acid sequence and the following Dayhoff sequences (allovomucoids, data incorporated herein by reference): IOVO_FRAER,IOVO_FRAAF, IOVO_FRACO, IOVO_CYRMO, IOVO_STRCA, H61492, C61589,IOVO_POLPL, D61589, and IOVO_TURME.

Example 25 Isolation of cDNA Clones Encoding Human PRO1550 Polypeptides[UNQ762]

Use of the signal sequence algorithm described in Example 3 aboveallowed identification of an EST sequence from the Merck database,designated CELT15B7_(—)12, also referred herein as “DNA10022”. This ESTsequence was then compared to a variety of expressed sequence tag (EST)databases which included public and proprietary EST databases (e.g.,GenBank and LIFESEQ®) to identify existing homologies. The homologysearch was performed using the computer program BLAST or BLAST2 (Altshulet al., Methods in Enzymology 266:460-480 (1996)). Those comparisonsresulting in a BLAST score of 70 (or in some cases 90) or greater thatdid not encode known proteins were clustered and assembled into aconsensus DNA sequence with the program “phrap” (Phil Green, Universityof Washington, Seattle, Wash.). The consensus sequence obtainedtherefrom is herein designated “DNA55708”.

In light of the sequence homology between the DNA55708 sequence and asequence contained within Incyte EST no. 3411659, the EST clone 3411659was purchased and the cDNA insert was obtained and sequenced in itsentirety. The sequence of this cDNA insert is shown in FIG. 45 and isherein designated as “DNA76393-1664”.

The full length clone shown in FIG. 45 contained a single open readingframe with an apparent translational initiation site at nucleotidepositions 138 to 140 and ending at the stop codon found at nucleotidepositions 867 to 869 (FIG. 45; SEQ ID NO:45). The predicted polypeptideprecursor (FIG. 46, SEQ ID NO:46) is 243 amino acids long. Otherfeatures of the PRO1550 protein include: a signal sequence at aboutamino acids 1-30; a hydrophobic domain at about amino acids 195-217; anda potential N-glycosylation site at about amino acids 186-189. PRO1550has a calculated molecular weight of approximately 26,266 daltons and anestimated pI of approximately 8.43. Clone DNA76393-1664 was depositedwith the ATCC on Oct. 6, 1998, and is assigned ATCC deposit no. 203323.

An analysis of the Dayhoff database (version 35.45 SwissProt 35), usinga WU-BLAST2 sequence alignment analysis of the full-length sequenceshown in FIG. 46 (SEQ ID NO:46), revealed some homology between thePRO1550 amino acid sequence and the following Dayhoff sequences:CELF59E12_(—)11; CA24_ASCSU; AF018082_(—)1; CA13_BOVIN; CA54_HUMAN;CA34_HUMAN; HUMCOL7A1X_(—)1; P_W09643; AF053538_(—)1; andHSEMCXIV2_(—)1.

Example 26 Isolation of cDNA Clones Encoding Human PRO1571 Polypeptides[UNQ777]

A consensus DNA sequence was assembled relative to other EST sequencesusing phrap as described in Example 1 above. This consensus sequence isherein designated DNA69559. Based on homology observed between theDNA69559 consensus sequence and an EST sequence contained within theIncyte EST clone no. 3140760, Incyte EST clone no. 3140760 was purchasedand the cDNA insert was obtained and sequenced. The sequence of thiscDNA insert is shown in FIG. 47 and is herein designated asDNA73730-1679.

Clone DNA73730-1679 contains a single open reading frame with anapparent translational initiation site at nucleotide positions 90-92 andending at the stop codon at nucleotide positions 807-809 (FIG. 47; SEQID NO:47). The predicted polypeptide precursor is 239 amino acids long(FIG. 48). The full-length PRO1571 protein shown in FIG. 48 has anestimated molecular weight of about 25,699 daltons and a pI of about8.99. Analysis of the full-length PRO1571 sequence shown in FIG. 48 (SEQID NO:48) evidences the presence of the following: a signal peptide fromabout amino acid 1 to about amino acid 21 and transmembrane domains fromabout amino acid 82 to about amino acid 103, from about amino acid 115to about amino acid 141 and from about amino acid 160 to about aminoacid 182. Clone DNA73730-1679 has been deposited with ATCC on Oct. 6,1998 and is assigned ATCC deposit no. 203320.

An analysis of the Dayhoff database (version 35.45 SwissProt 35), usinga WU-BLAST2 sequence alignment analysis of the full-length sequenceshown in FIG. 48 (SEQ ID NO:48), evidenced significant homology betweenthe PRO1571 amino acid sequence and the following Dayhoff sequences:AF072128_(—)1, AB000712_(—)1, AB000714_(—)1, AF007189_(—)1,AF000959_(—)1, AF068863_(—)1, P_W15288, PM22_HUMAN, P_R30056 andLSU46824_(—)1.

Example 27 Isolation of cDNA Clones Encoding Human PRO1572 Polypeptides[UNQ778]

Using the method described in Example 1 above, a consensus sequence wasobtained. The consensus sequence is designated herein “DNA69560”. Basedon the DNA69560 consensus sequence and other information providedherein, a clone including another EST (Incyte DNA3051424) from theassembly was purchased and sequenced.

The entire coding sequence of PRO1573 is included in FIG. 49 (SEQ IDNO:49). Clone DNA73734-1680 contains a single open reading frame with anapparent translational initiation site at nucleotide positions 90-92 andan apparent stop codon at nucleotide positions 873-875. The predictedpolypeptide precursor is 261 amino acids long. The signal peptide is atabout amino acids 1-23 and the transmembrane domains are at about aminoacids 81-100, 121-141, and 173-194 of SEQ ID NO: 50. One or more of thetransmembrane domains can be deleted or inactivated. The locations of aN-glycosylation site, N-myristoylation sites, a tyrosine kinasephosphorylation site and a prokaryotic membrane lipoprotein lipidattachment site are indicated in FIG. 50. Clone DNA73734-1680 has beendeposited with the ATCC on Oct. 20, 1998 and is assigned ATCC depositno. 203363. The full-length PRO1572 protein shown in FIG. 50 has anestimated molecular weight of about 27,856 daltons and a pI of about8.5.

An analysis of the Dayhoff database (version 35.45 SwissProt 35), usinga WU-BLAST2 sequence alignment analysis of the full-length sequenceshown in FIG. 50 (SEQ ID NO:50), revealed sequence identity between thePRO1572 amino acid sequence and the following Dayhoff sequences(incorporated herein): AF072127_(—)1, HSU89916_(—)1, AB000713_(—)1,AB000714_(—)1, AB000712_(—)1, AF000959_(—)1, AF072128_(—)1,AF068863_(—)1, P_W29881, and P_W58869.

Example 28 Isolation of cDNA Clones Encoding Human PRO1759 Polypeptides[UNQ832]

Use of the signal sequence algorithm described in Example 3 aboveallowed identification of an EST cluster sequence from the Incytedatabase, designated DNA10571. This EST cluster sequence was thencompared to a variety of expressed sequence tag (EST) databases whichincluded public EST databases (e.g., GenBank) and a proprietary EST DNAdatabase (LIFESEQ®, Incyte Pharmaceuticals, Palo Alto, Calif.) toidentify existing homologies. One or more of the ESTs was derived frompooled eosinophils of allergic asthmatic patients. The homology searchwas performed using the computer program BLAST or BLAST2 (Altshul etal., Methods in Enzymology 266:460-480 (1996)). Those comparisonsresulting in a BLAST score of 70 (or in some cases 90) or greater thatdid not encode known proteins were clustered and assembled into aconsensus DNA sequence with the program “phrap” (Phil Green, Universityof Washington, Seattle, Wash.). The consensus sequence obtainedtherefrom is herein designated DNA57313.

In light of the sequence homology between the DNA57313 sequence and theIncyte EST 2434255, the clone including this EST was purchased and thecDNA insert was obtained and sequenced. The sequence of this cDNA insertis shown in FIG. 51 and is herein designated as DNA76531-1701.

The full length clone shown in FIG. 51 contained a single open readingframe with an apparent translational initiation site at nucleotidepositions 125-127 and ending at the stop codon found at nucleotidepositions 1475-1477 (FIG. 51; SEQ ID NO:51). The approximate locationsof the signal peptide and transmembrane domains are indicated in FIG.52, whereas the approximate locations for N-myristoylation sites, alipid attachment site, an amidation site and a kinase phosphorylationsite are indicated in FIG. 52. The predicted polypeptide precursor (FIG.52, SEQ ID NO: 52) is 450 amino acids long. PRO1759 has a calculatedmolecular weight of approximately 49,765 daltons and an estimated pI ofapproximately 8.14. Clone DNA76531-1701 was deposited with the ATCC onNov. 17, 1998 and is assigned ATCC deposit no. 203465.

An analysis of the Dayhoff database (version 35.45 SwissProt 35), usinga WU-BLAST2 sequence alignment analysis of the full-length sequenceshown in FIG. 52 (SEQ ID NO:52), revealed sequence identity between thePRO1759 amino acid sequence and the following Dayhoff sequences:OPDE_PSEAE, TH11_TRYBB, S67684, RGT2_YEAST, S68362, ATSUGTRPR_(—)1,P_W17836 (Patent application WO9715668-A2), F69587, A48076, and A45611.

Example 29 Isolation of cDNA Clones Encoding Human PRO4341 Polypeptides[UNQ1895]

The extracellular domain (ECD) sequences (including the secretion signalsequence, if any) from about 950 known secreted proteins from theSwiss-Prot public database were used to search EST databases. The ESTdatabases included public EST databases (e.g., GenBank), and aproprietary EST database (LIFESEQ®, Incyte Pharmaceuticals, Palo Alto,Calif.). The search was performed using the computer program BLAST orBLAST2 [Altschul et al., Methods in Enzymology, 266:460-480 (1996)] as acomparison of the ECD protein sequences to a 6 frame translation of theEST sequences. Those comparisons resulting in a BLAST score of 70 (or insome cases, 90) or greater that did not encode known proteins wereclustered and assembled into consensus DNA sequences with the program“phrap” (Phil Green, University of Washington, Seattle, Wash.).

A consensus DNA sequence encoding PRO4341 was assembled relative toother EST sequences using phrap. This consensus sequence is designatedherein “DNA45433”.

Based on the DNA45433 consensus sequence oligonucleotides weresynthesized: 1) to identify by PCR a cDNA library that contained thesequence of interest, and 2) for use as probes to isolate a clone of thefull-length coding sequence for PRO4341. Forward and reverse PCR primersgenerally range from 20 to 30 nucleotides and are often designed to givea PCR product of about 100-1000 bp in length. The probe sequences aretypically 40-55 bp in length. In some cases, additional oligonucleotidesare synthesized when the consensus sequence is greater than about 1-1.5kbp. In order to screen several libraries for a full-length clone, DNAfrom the libraries was screened by PCR amplification, as per Ausubel etal., Current Protocols in Molecular Biology, supra, with the PCR primerpair. A positive library was then used to isolate clones encoding thegene of interest using the probe oligonucleotide and one of the primerpairs.

PCR primers (forward and reverse) were synthesized:

forward PCR primer 5′TTCTCTGGCCGACGCTGTGAGG3′; (SEQ ID NO: 152) andreverse PCR primer 5′GCCATAAGGGCATTGCACACAAAGG3′. (SEQ ID NO: 153)

Additionally, a synthetic oligonucleotide hybridization probe wasconstructed from the consensus 45433 sequence which had the followingnucleotide sequence:

hybridization probe (SEQ ID NO: 154)5′AGTCCCTGCTTCAACAGGGCCACCTGCTACCCGACCTCTCCAC3′.

In order to screen several libraries for a source of a full-lengthclone, DNA from the libraries was screened by PCR amplification with thePCR primer pair identified above. A positive library was then used toisolate clones encoding the PRO4341 gene using the probe oligonucleotideand one of the PCR primers.

RNA for construction of the cDNA libraries was isolated from human fetallung. The cDNA libraries used to isolate the cDNA clones wereconstructed by standard methods using commercially available reagentssuch as those from Invitrogen, San Diego, Calif. The cDNA was primedwith oligo dT containing a NotI site, linked with blunt to SalIhemikinased adaptors, cleaved with NotI, sized appropriately by gelelectrophoresis, and cloned in a defined orientation into a suitablecloning vector (such as pRKB or pRKD; pRK5B is a precursor of pRK5D thatdoes not contain the SfiI site; see, Holmes et al., Science,253:1278-1280 (1991)) in the unique XhoI and NotI sites.

DNA sequencing of the clones isolated as described above gave thefull-length DNA sequence for PRO4341 (designated herein as DNA81761-2583[FIG. 57, SEQ ID NO:57]; and the derived protein sequence for PRO4341.

The entire coding sequence of PRO4341 is shown in FIG. 57 (SEQ IDNO:57). Clone DNA81761-2583 contains a single open reading frame with anapparent translational initiation site at nucleotide positions 36-38,and an apparent stop codon at nucleotide positions 2091-2093. Thepredicted polypeptide precursor is 685 amino acids long. CloneDNA81761-2583 (UNQ1895), designated as DNA81761-2583 has been depositedwith ATCC on Mar. 23, 1999 and is assigned ATCC deposit no. 203862. Thefull-length PRO4341 protein shown in FIG. 58 has an estimated molecularweight of about 74605 daltons and a pI of about 6.89.

An analysis of the Dayhoff database (version 35.45 SwissProt 35), usinga WU-BLAST2 sequence alignment analysis of the full-length sequenceshown in FIG. 58 (SEQ ID NO: 58), revealed homology between the PRO4341amino acid sequence and the following Dayhoff sequences (sequences andrelated text incorporated herein): P_W11719, I50719, P_WO0876,DLL1_HUMAN, P_W18348, AF030031_(—)1, AF020201_(—)1, AF028593_(—)1,P_WO5833 and CRB_DROME. Therefore, it is believed that PRO4341 isrelated to Delta and useful in the treatment of cancer, wound repair,differentiation disorders or in assays to development compounds whichare useful in such treatments.

Example 30 Isolation of cDNA Clones Encoding Human PRO4348 Polypeptides[UNQ1902]

The extracellular domain (ECD) sequences (including the secretion signalsequence, if any) from about 950 known secreted proteins from theSwiss-Prot public database were used to search EST databases. The ESTdatabases included public EST databases (e.g., GenBank), and aproprietary EST database (LIFESEQ®, Incyte Pharmaceuticals, Palo Alto,Calif.). The search was performed using the computer program BLAST orBLAST2 [Altschul et al., Methods in Enzymology, 266:460-480 (1996)] as acomparison of the ECD protein sequences to a 6 frame translation of theEST sequences. Those comparisons resulting in a BLAST score of 70 (or insome cases, 90) or greater that did not encode known proteins wereclustered and assembled into consensus DNA sequences with the program“phrap” (Phil Green, University of Washington, Seattle, Wash.).

A consensus DNA sequence encoding PRO4348 was assembled relative toother EST sequences using phrap. This consensus sequence is designatedherein “DNA77500”.

Based on the DNA77500 consensus sequence, DNA92232-2589 was identified.DNA sequencing gave the full-length DNA sequence for PRO4348 (designatedherein as DNA92232-2589 [FIG. 59, SEQ ID NO:59]; and the derived proteinsequence for PRO4348.

The entire coding sequence of PRO4348 is shown in FIG. 59 (SEQ IDNO:59). Clone DNA92232-2589 contains a single open reading frame with anapparent translational initiation site at nucleotide positions 57-59,and an apparent stop codon at nucleotide positions 789-791 of SEQ IDNO:59. The predicted polypeptide precursor is 244 amino acids long.Clone DNA92232-2589 (UNQ1902), designated as DNA92232-2589 has beendeposited with ATCC ON Mar. 30, 1999 and is assigned ATCC deposit no.203895. The full-length PRO4348 protein shown in FIG. 60 has anestimated molecular weight of about 28319 daltons and a pI of about8.78.

An analysis of the Dayhoff database (version 35.45 SwissProt 35), usinga WU-BLAST2 sequence alignment analysis of the full-length sequenceshown in FIG. 60 (SEQ ID NO:60), revealed homology between the PRO4348amino acid sequence and the following Dayhoff sequences: D70554, D69267,YH09_YEAST, D71620, AB019196_(—)1, F71102, COQ5_YEAST, BIOC_SERMA,S61202, and PMTA_RHOSH.

Example 31 Isolation of cDNA Clones Encoding Human PRO4369 Polypeptides[UNQ1911]

DNA92289-2598 was identified by applying a proprietary signal sequencefinding algorithm developed by Genentech, Inc. (South San Francisco,Calif.) upon ESTs as well as clustered and assembled EST fragments frompublic (e.g., GenBank) and/or private (LIFESEQ®, Incyte Pharmaceuticals,Inc., Palo Alto, Calif.) databases. The signal sequence algorithmcomputes a secretion signal score based on the character of the DNAnucleotides surrounding the first and optionally the second methioninecodon(s) (ATG) at the 5′-end of the sequence or sequence fragment underconsideration. The nucleotides following the first ATG must code for atleast 35 unambiguous amino acids without any stop codons. If the firstATG has the required amino acids, the second is not examined. If neithermeets the requirement, the candidate sequence is not scored. In order todetermine whether the EST sequence contains an authentic signalsequence, the DNA and corresponding amino acid sequences surrounding theATG codon are scored using a set of seven sensors (evaluationparameters) known to be associated with secretion signals.

Use of the above described signal sequence algorithm allowedidentification of an EST cluster sequence from the Incyte database. ThisEST cluster sequence was then compared to a variety of expressedsequence tag (EST) databases which included public EST databases (e.g.,GenBank) and a proprietary EST DNA database (LIFESEQ®, IncytePharmaceuticals, Palo Alto, Calif.) to identify existing homologies. Thehomology search was performed using the computer program BLAST or BLAST2(Altshul et al., Methods in Enzymology 266:460-480 (1996)). Thosecomparisons resulting in a BLAST score of 70 (or in some cases 90) orgreater that did not encode known proteins were clustered and assembledinto a consensus DNA sequence with the program “phrap” (Phil Green,University of Washington, Seattle, Wash.). The consensus sequenceobtained therefrom is herein designated DNA75180. In light of DNA75180,DNA92289 was identified.

The full length clone shown in FIG. 61 contained a single open readingframe with an apparent translational initiation site at nucleotidepositions 74-76 and ending at the stop codon found at nucleotidepositions 776-778 (FIG. 61; SEQ ID NO:61). The predicted polypeptideprecursor (FIG. 62, SEQ ID NO:62) is 234 amino acids long. PRO4369 has acalculated molecular weight of approximately 26077 daltons and anestimated pI of approximately 8.13.

An analysis of the Dayhoff database (version 35.45 SwissProt 35), usinga WU-BLAST2 sequence alignment analysis of the full-length sequenceshown in FIG. 62 (SEQ ID NO: 62), revealed homology between the PRO4369amino acid sequence and the following Dayhoff sequences (sequences andrelated text incorporated herein): Y081_HUMAN, NUCL_CHICK, S64439,YG3A_YEAST, CELF12F3_(—)3, ATGELSOLI_(—)1, S55395, NFM_RABIT,PFAHSP86B_(—)1 and NPM_XENLA.

Clone DNA92289-2598 (UNQ1911), designated as DNA92289-2598 was depositedwith the ATCC on May 25, 1999 and is assigned ATCC deposit no. PTA-131.

Example 32 Isolation of cDNA Clones Encoding Human PRO4381 Polypeptides[UNQ1916]

DNA92225-2603 was identified by applying a proprietary signal sequencefinding algorithm developed by Genentech, Inc. (South San Francisco,Calif.) upon ESTs as well as clustered and assembled EST fragments frompublic (e.g., GenBank) and/or private (LIFESEQ®, Incyte Pharmaceuticals,Inc., Palo Alto, Calif.) databases. The signal sequence algorithmcomputes a secretion signal score based on the character of the DNAnucleotides surrounding the first and optionally the second methioninecodon(s) (ATG) at the 5′-end of the sequence or sequence fragment underconsideration. The nucleotides following the first ATG must code for atleast 35 unambiguous amino acids without any stop codons. If the firstATG has the required amino acids, the second is not examined. If neithermeets the requirement, the candidate sequence is not scored. In order todetermine whether the EST sequence contains an authentic signalsequence, the DNA and corresponding amino acid sequences surrounding theATG codon are scored using a set of seven sensors (evaluationparameters) known to be associated with secretion signals.

Use of the above described signal sequence algorithm allowedidentification of an EST sequence from the Incyte database. This ESTsequence was then compared to a variety of expressed sequence tag (EST)databases which included public EST databases (e.g., GenBank) and aproprietary EST DNA database (LIFESEQ®, Incyte Pharmaceuticals, PaloAlto, Calif.) to identify existing homologies. The homology search wasperformed using the computer program BLAST or BLAST2 (Altshul et al.,Methods in Enzymology 266:460-480 (1996)). Those comparisons resultingin a BLAST score of 70 (or in some cases 90) or greater that did notencode known proteins were clustered and assembled into a consensus DNAsequence with the program “phrap” (Phil Green, University of Washington,Seattle, Wash.). One or more of the ESTs used in the assembly wasderived from a thymus tissue library. The consensus sequence obtainedtherefrom is herein designated DNA79136. In light of the DNA79136sequence DNA92225-2603 was identified and sequenced.

The full length clone shown in FIG. 63 contained a single open readingframe with an apparent translational initiation site at nucleotidepositions 145-147 and ending at the stop codon found at nucleotidepositions 460-462 (FIG. 63; SEQ ID NO: 63). The predicted polypeptideprecursor (FIG. 64, SEQ ID NO: 64) is 105 amino acids long. PRO4381 hasa calculated molecular weight of approximately 10803 daltons and anestimated pI of approximately 7.2.

An analysis of the Dayhoff database (version 35.45 SwissProt 35), usinga WU-BLAST2 sequence alignment analysis of the full-length sequenceshown in FIG. 64 (SEQ ID NO: 64), revealed homology between the PRO4381amino acid sequence and the following Dayhoff sequences (sequences andrelated text incorporated herein): A2AC_CAVPO, P102 KB_(—)39, S7123,I54343, HSL25A3_(—)1, C71466, P_R62382, S76774, HS0934, and A64763.

Clone DNA92225-2603 (UNQ1916), designated as DNA92225-2603 was depositedwith the ATCC on Apr. 20, 1999 and is assigned ATCC deposit no. 203950.

Example 33 Isolation of cDNA Clones Encoding Human PRO4407 Polypeptides[UNQ1932]

DNA92264-2616 was identified by applying a proprietary signal sequencefinding algorithm developed by Genentech, Inc. (South San Francisco,Calif.) upon ESTs as well as clustered and assembled EST fragments frompublic (e.g., GenBank) and/or private (LIFESEQ®, Incyte Pharmaceuticals,Inc., Palo Alto, Calif.) databases. The signal sequence algorithmcomputes a secretion signal score based on the character of the DNAnucleotides surrounding the first and optionally the second methioninecodon(s) (ATG) at the 5′-end of the sequence or sequence fragment underconsideration. The nucleotides following the first ATG must code for atleast 35 unambiguous amino acids without any stop codons. If the firstATG has the required amino acids, the second is not examined. If neithermeets the requirement, the candidate sequence is not scored. In order todetermine whether the EST sequence contains an authentic signalsequence, the DNA and corresponding amino acid sequences surrounding theATG codon are scored using a set of seven sensors (evaluationparameters) known to be associated with secretion signals.

Use of the above described signal sequence algorithm allowedidentification of an EST cluster sequence from the Incyte database. ThisEST cluster sequence was then compared to a variety of expressedsequence tag (EST) databases which included public EST databases (e.g.,GenBank) and a proprietary EST DNA database (LIFESEQ®, IncytePharmaceuticals, Palo Alto, Calif.) to identify existing homologies. Thehomology search was performed using the computer program BLAST or BLAST2(Altshul et al., Methods in Enzymology 266:460-480 (1996)). Thosecomparisons resulting in a BLAST score of 70 (or in some cases 90) orgreater that did not encode known proteins were clustered and assembledinto a consensus DNA sequence with the program “phrap” (Phil Green,University of Washington, Seattle, Wash.). Based upon the clustersequence and the sequence alignments, DNA92264-2616 was identified andsequenced.

The full length clone shown in FIG. 65 contained a single open readingframe with an apparent translational initiation site at nucleotidepositions 109-111 and ending at the stop codon found at nucleotidepositions 757-759 (FIG. 65; SEQ ID NO: 65). The predicted polypeptideprecursor (FIG. 66, SEQ ID NO: 66) is 216 amino acids long. PRO4407 hasa calculated molecular weight of approximately 23729 daltons and anestimated pI of approximately 4.73.

An analysis of the Dayhoff database (version 35.45 SwissProt 35), usinga WU-BLAST2 sequence alignment analysis of the full-length sequenceshown in FIG. 66 (SEQ ID NO: 66), revealed homology between the PRO4407amino acid sequence and the following Dayhoff sequences: SC1E6_(—)12,D80003_(—)1, HMGA_SOYBN, DROTRO12_(—)1, HSU91934_(—)1, GEN14338,AF051945_(—)1, A45644, P_W60213, and P_W33807.

Clone DNA92264-2616 (UNQ1932), designated as DNA92264-2616 was depositedwith the ATCC on Apr. 27, 1999 and is assigned ATCC deposit no. 203969.

Example 34 Isolation of cDNA Clones Encoding Human PRO4425 Polypeptides[UNQ1942]

Use of the signal sequence algorithm described in Example 3 aboveallowed identification of an EST cluster sequence from the Incytedatabase. This EST cluster sequence was then compared to a variety ofexpressed sequence tag (EST) databases which included public ESTdatabases (e.g., GenBank) and a proprietary EST DNA database (LIFESEQ®,Incyte Pharmaceuticals, Palo Alto, Calif.) to identify existinghomologies. The homology search was performed using the computer programBLAST or BLAST2 (Altshul et al., Methods in Enzymology 266:460-480(1996)). Those comparisons resulting in a BLAST score of 70 (or in somecases 90) or greater that did not encode known proteins were clusteredand assembled into a consensus DNA sequence with the program “phrap”(Phil Green, University of Washington, Seattle, Wash.). The consensussequence obtained therefrom is herein designated DNA81099.

In light of an observed sequence homology between the DNA81099 sequenceand an EST sequence contained within the EST clone no. AA448744, the ESTclone AA448744 was purchased from Merck and the cDNA insert was obtainedand sequenced. The sequence of this cDNA insert is herein designated asDNA93011-2637.

The full length clone shown in FIG. 67 contained a single open readingframe with an apparent translational initiation site at nucleotidepositions 27-29 and ending at the stop codon found at nucleotidepositions 435-437 (FIG. 67; SEQ ID NO:67). The predicted polypeptideprecursor (FIG. 68, SEQ ID NO:68) is 136 amino acids long. PRO4425 has acalculated molecular weight of approximately 15,577 daltons and anestimated pI of approximately 8.88.

An analysis of the Dayhoff database (version 35.45 SwissProt 35), usinga WU-BLAST2 sequence alignment analysis of the full-length sequenceshown in FIG. 68 (SEQ ID NO:68), revealed homology between the PRO4425amino acid sequence and the following Dayhoff sequences: HGS_RE295,S44655, YOJ8_CAEEL, VBR1_CLVK, P_R39520, P_R65332, P_R39388, TGL4_HUMAN,YKAB_CAEEL, and S71105.

Clone DNA93011-2637 was deposited with the ATCC on May 4, 1999 and isassigned ATCC deposit no. 20-PTA.

Example 35 Isolation of cDNA Clones Encoding Human PRO4985 Polypeptides[UNQ2426]

The extracellular domain (ECD) sequences (including the secretion signalsequence, if any) from about 950 known secreted proteins from theSwiss-Prot public database were used to search EST databases. The ESTdatabases included a proprietary EST database (LIFESEQ®, IncytePharmaceuticals, Palo Alto, Calif.). The search was performed using thecomputer program BLAST or BLAST2 [Altschul et al., Methods inEnzymology, 266:460-480 (1996)] as a comparison of the ECD proteinsequences to a 6 frame translation of the EST sequences. Thosecomparisons resulting in a BLAST score of 70 (or in some cases, 90) orgreater that did not encode known proteins were clustered and assembledinto consensus DNA sequences with the program “phrap” (Phil Green,University of Washington, Seattle, Wash.).

A consensus DNA sequence was assembled relative to other EST sequencesusing phrap as described above. This consensus sequence is hereindesignated DNA42819. In some cases, the DNA42819 consensus sequencederives from an intermediate consensus DNA sequence which was extendedusing repeated cycles of BLAST and phrap to extend that intermediateconsensus sequence as far as possible using the sources of EST sequencesdiscussed above.

Based on the DNA42819 consensus sequence oligonucleotides weresynthesized: 1) to identify by PCR a cDNA library that contained thesequence of interest, and 2) for use as probes to isolate a clone of thefull-length coding sequence for PRO4985. Forward and reverse PCR primersgenerally range from 20 to 30 nucleotides and are often designed to givea PCR product of about 100-1000 bp in length. The probe sequences aretypically 40-55 bp in length. In some cases, additional oligonucleotidesare synthesized when the consensus sequence is greater than about 1-1.5kbp. In order to screen several libraries for a full-length clone, DNAfrom the libraries was screened by PCR amplification, as per Ausubel etal., Current Protocols in Molecular Biology, supra, with the PCR primerpair. A positive library was then used to isolate clones encoding thegene of interest using the probe oligonucleotide and one of the primerpairs.

PCR primers (forward and reverse) were synthesized:

forward PCR primer 5′-TGTCCTCTATTGGAGAACCACAGCC-3′ (SEQ ID NO: 155)reverse PCR primer 5′-TAAAAGTTGGCTGGGCAAAGTTTGC-3′ (SEQ ID NO: 156)Additionally, a synthetic oligonucleotide hybridization probe wasconstructed from the consensus DNA42819 sequence which had the followingnucleotide sequence

hybridization probe (SEQ ID NO: 157)5′-CTCAGTATGGACCAAAGTACCCAAGCCTGTGCTGGTGAGAAACATTG GCA-3′

RNA for construction of the cDNA libraries was isolated from humanthyroid tissue. The cDNA libraries used to isolate the cDNA clones wereconstructed by standard methods using commercially available reagentssuch as those from Invitrogen, San Diego, Calif. The cDNA was primedwith oligo dT containing a NotI site, linked with blunt to SalIhemikinased adaptors, cleaved with NotI, sized appropriately by gelelectrophoresis, and cloned in a defined orientation into a suitablecloning vector (such as pRKB or pRKD; pRK5B is a precursor of pRK5D thatdoes not contain the SfiI site; see, Holmes et al., Science,253:1278-1280 (1991)) in the unique XhoI and NotI sites.

DNA sequencing of the clones isolated as described above gave thefull-length DNA sequence for a full-length PRO4985 polypeptide(designated herein as DNA59770-2652 [FIG. 69, SEQ ID NO: 69]) and thederived protein sequence for that PRO4985 polypeptide.

The full length clone identified above contained a single open readingframe with an apparent translational initiation site at nucleotidepositions 133-135 and a stop signal at nucleotide positions 3172-3174(FIG. 69, SEQ ID NO:69). The predicted polypeptide precursor is 1013amino acids long, has a calculated molecular weight of approximately111,348.50 daltons and an estimated pI of approximately 6.34. Analysisof the full-length PRO4985 sequence shown in FIG. 70 (SEQ ID NO:70)evidences the presence of a variety of important polypeptide domains asshown in FIG. 70, wherein the locations given for those importantpolypeptide domains are approximate as described above. CloneDNA59770-2652 has been deposited with ATCC on Jul. 27, 1999 and isassigned ATCC Deposit No. PTA-427.

An analysis of the Dayhoff database (version 35.45 SwissProt 35), usingthe ALIGN-2 sequence alignment analysis of the full-length sequenceshown in FIG. 70 (SEQ ID NO:70), evidenced sequence identity between thePRO4985 amino acid sequence and the following Dayhoff sequences:CEF58E6_(—)3; XELERTK_(—)1; CELW02C12_(—)2; I49071; I48653; EPB3_MOUSE;EPB3_HUMAN; LMG1_DROME; CVU90226_(—)1; P_W57046.

Example 36 Isolation of cDNA Clones Encoding Human PRO4989 Polypeptides[UNQ2429]

The extracellular domain (ECD) sequences (including the secretion signalsequence, if any) from about 950 known secreted proteins from theSwiss-Prot public database were used to search EST databases. The ESTdatabases included (1) public EST databases (e.g., Merck/WashingtonUniversity), (2) a proprietary EST database (LIFESEQ®, IncytePharmaceuticals, Palo Alto, Calif.), and (3) a proprietary EST databasefrom Genentech. The search was performed using the computer programBLAST or BLAST2 [Altschul et al., Methods in Enzymology, 266:460-480(1996)] as a comparison of the ECD protein sequences to a 6 frametranslation of the EST sequences. Those comparisons resulting in a BLASTscore of 70 (or in some cases, 90) or greater that did not encode knownproteins were clustered and assembled into consensus DNA sequences withthe program “phrap” (Phil Green, University of Washington, Seattle,Wash.).

A consensus DNA sequence was assembled relative to other EST sequencesusing phrap as described above. This consensus sequence is hereindesignated DNA54206. In some cases, the DNA54206 consensus sequencederives from an intermediate consensus DNA sequence which was extendedusing repeated cycles of BLAST and phrap to extend that intermediateconsensus sequence as far as possible using the sources of EST sequencesdiscussed above.

Based on the DNA54206 consensus sequence oligonucleotides weresynthesized: 1) to identify by PCR a cDNA library that contained thesequence of interest, and 2) for use as probes to isolate a clone of thefull-length coding sequence for PRO4989. Forward and reverse PCR primersgenerally range from 20 to 30 nucleotides and are often designed to givea PCR product of about 100-1000 bp in length. The probe sequences aretypically 40-55 bp in length. In some cases, additional oligonucleotidesare synthesized when the consensus sequence is greater than about 1-1.5kbp. In order to screen several libraries for a full-length clone, DNAfrom the libraries was screened by PCR amplification, as per Ausubel etal., Current Protocols in Molecular Biology, supra, with the PCR primerpair. A positive library was then used to isolate clones encoding thegene of interest using the probe oligonucleotide and one of the primerpairs.

PCR primers (forward and reverse) were synthesized:

forward PCR primer 5′-CAAGGTCCTGCGGAATGTCTCTGG-3′ (SEQ ID NO: 158)reverse PCR primer 5′-GGGAAGTCCTGGAACTGGTTCCGG-3′ (SEQ ID NO: 159)Additionally, a synthetic oligonucleotide hybridization probe wasconstructed from the consensus DNA54206 sequence which had the followingnucleotide sequence

hybridization probe (SEQ ID NO: 160)5′-CCTCATCACCCTGGCTAACAACGAGCTTAAGTCCCTCACCAGCAA G-3′

RNA for construction of the cDNA libraries was isolated from humantestis tissue. The cDNA libraries used to isolate the cDNA clones wereconstructed by standard methods using commercially available reagentssuch as those from Invitrogen, San Diego, Calif. The cDNA was primedwith oligo dT containing a NotI site, linked with blunt to SalIhemikinased adaptors, cleaved with NotI, sized appropriately by gelelectrophoresis, and cloned in a defined orientation into a suitablecloning vector (such as pRKB or pRKD; pRK5B is a precursor of pRK5D thatdoes not contain the SfiI site; see, Holmes et al., Science,253:1278-1280 (1991)) in the unique XhoI and NotI sites.

DNA sequencing of the clones isolated as described above gave thefull-length DNA sequence for a full-length PRO4989 polypeptide(designated herein as DNA80135-2655 [FIG. 71, SEQ ID NO: 71]) and thederived protein sequence for that PRO4989 polypeptide.

The full length clone identified above contained a single open readingframe with an apparent translational initiation site at nucleotidepositions 223-225 and a stop signal at nucleotide positions 775-777(FIG. 71, SEQ ID NO:71). The predicted polypeptide precursor is 184amino acids long, has a calculated molecular weight of approximately20,509 daltons and an estimated pI of approximately 6.47. Analysis ofthe full-length PRO4989 sequence shown in FIG. 72 (SEQ ID NO:72)evidences the presence of a variety of important polypeptide domains asshown in FIG. 72, wherein the locations given for those importantpolypeptide domains are approximate as described above. Clone DNA80135has been deposited with ATCC on Jun. 15, 1999 and is assigned ATCCdeposit no. PTA-234.

An analysis of the Dayhoff database (version 35.45 SwissProt 35), usingthe ALIGN-2 sequence alignment analysis of the full-length sequenceshown in FIG. 72 (SEQ ID NO:72), evidenced sequence identity between thePRO4989-amino acid sequence and the following Dayhoff sequences:DDU82512_(—)1; AF061443_(—)1; AF054827_(—)1; AF068919_(—)1;AB016816_(—)1; ATY16046_(—)1; AF068920_(—)1; AF054828_(—)1; CYAA_YEAST;and CYAA_SCHPO.

Example 37 Isolation of cDNA Clones Encoding Human PRO5737 Polypeptides[UNQ2456]

An expressed sequence tag (EST) DNA database (LIFESEQ®, IncytePharmaceuticals, Palo Alto, Calif.) was searched with a humaninterleukin-1 receptor antagonist (hIL-1Ra) sequence, and an ESTsequence, designated herein as 1433156 was identified, which showedhomology with the hIL-1Ra known protein. EST clone 1433156 was purchasedfrom Incyte Pharmaceuticals (Palo Alto, Calif.) and the cDNA insert wasobtained and sequenced in its entirety, giving the DNA92929-2534sequence.

The entire nucleotide sequence of DNA92929-2534 is shown in FIG. 73 (SEQID NO:73). Clone DNA92929-2534 contains a single open reading frame withan apparent translational initiation site at nucleotide positions 96-98and a stop codon at nucleotide positions 498-500 (FIG. 73; SEQ IDNO:73). The predicted polypeptide precursor (hIL-1Ra2) is 134 aminoacids long. The putative signal sequence extends from amino acidpositions 1-17. Clone DNA92929-2534 was deposited with ATCC on Jan. 12,1999 and was assigned ATCC deposit no. 203586. The full-length hIL-Ira2protein shown in FIG. 74 has an estimated molecular weight of about14,927 daltons and a pI of about 4.8.

Based on a BLAST and FastA sequence alignment analysis (using theALIGN-2 computer program) of the full-length sequence, hIL-1Ra2 (FIG.74, SEQ ID NO:74) shows significant amino acid sequence identity tohIL-1R protein. hIL-1Ra2 is believed to be a splice variant of hIL-1R.

Example 38 Isolation of cDNA Clones Encoding Human PRO5800 Polypeptides[UNQ2500]

The extracellular domain (ECD) sequences (including the secretion signalsequence, if any) from about 950 known secreted proteins from theSwiss-Prot public database were used to search EST databases. The ESTdatabases included public EST databases (e.g., GenBank). The search wasperformed using the computer program BLAST or BLAST2 [Altschul et al.,Methods in Enzymology, 266:460-480 (1996)] as a comparison of the ECDprotein sequences to a 6 frame translation of the EST sequences. Thosecomparisons resulting in a BLAST score of 70 (or in some cases, 90) orgreater that did not encode known proteins were clustered and assembledinto consensus DNA sequences with the program “phrap” (Phil Green,University of Washington, Seattle, Wash.).

A consensus DNA sequence was assembled relative to other EST sequencesusing phrap as described above. This consensus sequence is hereindesignated DNA102836. In some cases, the consensus sequence derives froman intermediate consensus DNA sequence which was extended using repeatedcycles of BLAST and phrap to extend that intermediate consensus sequenceas far as possible using the sources of EST sequences discussed above.

Based on the DNA102836 consensus sequence oligonucleotides weresynthesized: 1) to identify by PCR a cDNA library that contained thesequence of interest, and 2) for use as probes to isolate a clone of thefull-length coding sequence for PRO5800. Forward and reverse PCR primersgenerally range from 20 to 30 nucleotides and are often designed to givea PCR product of about 100-1000 bp in length. The probe sequences aretypically 40-55 bp in length. In some cases, additional oligonucleotidesare synthesized when the consensus sequence is greater than about 1-1.5kbp. In order to screen several libraries for a full-length clone, DNAfrom the libraries was screened by PCR amplification, as per Ausubel etal., Current Protocols in Molecular Biology, supra, with the PCR primerpair. A positive library was then used to isolate clones encoding thegene of interest using the probe oligonucleotide and one of the primerpairs.

PCR primers (forward and reverse) were synthesized:

forward PCR primer 1 (SEQ ID NO: 161) 5′-CAGCGAACCGGGTGCCGGGTC-3′forward PCR primer 2 (SEQ ID NO: 162) 5′-GAGCGACGAGCGCGCAGCGAAC-3′forward PCR primer 3 (SEQ ID NO: 163)5′-ATACTGCGATCGCTAAACCACCATGCGCCGCCGCCTGTGGCTG-3′ reverse PCR primer 1(SEQ ID NO: 164) 5′-GCCGGCCTCTCAGGGCCTCAG-3′ reverse PCR primer 2(SEQ ID NO: 165) 5′-CCCACGTGTACAGAGCGGATCTC-3′ reverse PCR primer 3(SEQ ID NO: 166) 5′-GAGACCAGGACGGGCAGGAAGTG-3′ reverse PCR primer 4(SEQ ID NO: 167) 5′-CAGGCACCTTGGGGAGCCGCC-3′ reverse PCR primer 5(SEQ ID NO: 168) 5′-CCCACGTGTACAGAGCGGATCTC-3′ reverse PCR primer 6(SEQ ID NO: 169) 5′-GAGACCAGGACGGGCAGGAAGTG-3′Additionally, a synthetic oligonucleotide hybridization probe wasconstructed from the consensus DNA102836 sequence which had thefollowing nucleotide sequence

hybridization probe (SEQ ID NO: 170)5′-CTCTACGGGTACTGCAGGTTCCGGGAGCGCATCGAAGAGAACGG-3′

RNA for construction of the cDNA libraries was isolated from human fetalliver tissue. The cDNA libraries used to isolate the cDNA clones wereconstructed by standard methods using commercially available reagentssuch as those from Invitrogen, San Diego, Calif. The cDNA was primedwith oligo dT containing a NotI site, linked with blunt to SalIhemikinased adaptors, cleaved with NotI, sized appropriately by gelelectrophoresis, and cloned in a defined orientation into a suitablecloning vector (such as pRKB or pRKD; pRK5B is a precursor of pRK5D thatdoes not contain the SfiI site; see, Holmes et al., Science,253:1278-1280 (1991)) in the unique XhoI and NotI sites.

DNA sequencing of the clones isolated as described above gave thefull-length DNA sequence for a full-length PRO5800 polypeptide(designated herein as DNA108912-2680 [FIG. 75, SEQ ID NO: 75]) and thederived protein sequence for that PRO5800 polypeptide.

The full length clone identified above contained a single open readingframe with an apparent translational initiation site at nucleotidepositions 7-9 and a stop signal at nucleotide positions 517-519 (FIG.75, SEQ ID NO:75). The predicted polypeptide precursor is 170 aminoacids long, has a calculated molecular weight of approximately 19,663daltons and an estimated pI of approximately 11.81. Analysis of thefull-length PRO5800 sequence shown in FIG. 76 (SEQ ID NO:76) evidencesthe presence of a variety of important polypeptide domains as shown inFIG. 76, wherein the locations given for those important polypeptidedomains are approximate as described above. Clone DNA108912-2680 hasbeen deposited with ATCC on May 25, 1999 and is assigned ATCC depositno. PTA-124.

An analysis of the Dayhoff database (version 35.45 SwissProt 35), usingthe ALIGN-2 sequence alignment analysis of the full-length sequenceshown in FIG. 76 (SEQ ID NO:76), evidenced sequence identity between thePRO5800 amino acid sequence and the following Dayhoff sequences:P_W52595, P_W57313, FGFA_HUMAN, P_W57264, FGFA_RAT, P_W52597,MMU94517_(—)1, FGFA_MOUSE, P_W57306 and D86333_(—)1.

Example 39 Isolation of cDNA Clones Encoding Human PRO5993 Polypeptides[UNQ2504]

The extracellular domain (ECD) sequences (including the secretion signalsequence, if any) from about 950 known secreted proteins from theSwiss-Prot public database were used to search EST databases. The ESTdatabases included (1) public EST databases (e.g., Merck/WashingtonUniversity), (2) a proprietary EST database (LIFESEQ®, IncytePharmaceuticals, Palo Alto, Calif.), (3) a proprietary EST database fromGenentech. The search was performed using the computer program BLAST orBLAST2 [Altschul et al., Methods in Enzymology, 266:460-480 (1996)] as acomparison of the ECD protein sequences to a 6 frame translation of theEST sequences. Those comparisons resulting in a BLAST score of 70 (or insome cases, 90) or greater that did not encode known proteins wereclustered and assembled into consensus DNA sequences with the program“phrap” (Phil Green, University of Washington, Seattle, Wash.).

This consensus sequence is herein designated DNA91365. In some cases,the DNA91365 consensus sequence derives from an intermediate consensusDNA sequence which was extended using repeated cycles of BLAST and phrapto extend that intermediate consensus sequence as far as possible usingthe sources of EST sequences discussed above.

Based on the DNA91365 sequence oligonucleotides were synthesized: 1) toidentify by PCR a cDNA library that contained the sequence of interest,and 2) for use as probes to isolate a clone of the full-length codingsequence for PRO5993. Forward and reverse PCR primers generally rangefrom 20 to 30 nucleotides and are often designed to give a PCR productof about 100-1000 bp in length. The probe sequences are typically 40-55bp in length. In some cases, additional oligonucleotides are synthesizedwhen the consensus sequence is greater than about 1-1.5 kbp. In order toscreen several libraries for a full-length clone, DNA from the librarieswas screened by PCR amplification, as per Ausubel et al., CurrentProtocols in Molecular Biology, supra, with the PCR primer pair. Apositive library was then used to isolate clones encoding the gene ofinterest using the probe oligonucleotide and one of the primer pairs.

PCR primers (forward and reverse) were synthesized:

forward PCR primer 5′CGACCCAAGCGGATCGAAGGTTC 3′ (SEQ ID NO: 171)reverse PCR primer 5′GTCACTTCCTGGCACCAGCTGCTC 3′ (SEQ ID NO: 172)Additionally, a synthetic oligonucleotide hybridization probe wasconstructed from the consensus DNA91365 sequence which had the followingnucleotide sequence

hybridization probe (SEQ ID NO: 173)5′GTTAGCAACTCTCTGGCAGCCTTTGCTTACATTAGAGACCACCCG 3′

RNA for construction of the cDNA libraries was isolated from humanaortic endothelial tissue. The cDNA libraries used to isolate the cDNAclones were constructed by standard methods using commercially availablereagents such as those from Invitrogen, San Diego, Calif. The cDNA wasprimed with oligo dT containing a NotI site, linked with blunt to SalIhemikinased adaptors, cleaved with NotI, sized appropriately by gelelectrophoresis, and cloned in a defined orientation into a suitablecloning vector (such as pRKB or pRKD; pRK5B is a precursor of pRK5D thatdoes not contain the SfiI site; see, Holmes et al., Science,253:1278-1280 (1991)) in the unique XhoI and NotI sites.

DNA sequencing of the clones isolated as described above gave thefull-length DNA sequence for a full-length PRO5993 polypeptide(designated herein as DNA100276-2684 [FIG. 77, SEQ ID NO: 77]) and thederived protein sequence for that PRO5993 polypeptide.

The full length clone identified above contained a single open readingframe with an apparent translational initiation site at nucleotidepositions 411-413 and a stop signal at nucleotide positions 1734-1736(FIG. 77, SEQ ID NO: 77). The predicted polypeptide precursor is 441amino acids long, has a calculated molecular weight of approximately49483 daltons and an estimated pI of approximately 6.91. Analysis of thefull-length PRO5993 sequence shown in FIG. 78 (SEQ ID NO: 78) evidencesthe presence of a variety of important polypeptide domains as shown inFIG. 78, wherein the locations given for those important polypeptidedomains are approximate as described above. Clone DNA100276-2684 hasbeen deposited with ATCC on Jul. 20, 1999 and is assigned ATCC DepositNo. PTA-380.

An analysis of the Dayhoff database (version 35.45 SwissProt 35), usingthe ALIGN-2 sequence alignment analysis of the full-length sequenceshown in FIG. 78 (SEQ ID NO: 78), evidenced sequence identity betweenthe PRO5993 amino acid sequence and the following Dayhoff sequences:CEF32A7_(—)4; CEF32A7_(—)3; LEG_ANTCR; AF081149_(—)1; P_W74585;HASA131581_(—)1; RNU72487_(—)1; AF111098_(—)1; P_W59050.

Example 40 Isolation of cDNA Clones Encoding Human PRO6017 Polypeptides[UNQ2524]

DNA96860-2700 was identified by applying a proprietary signal sequencefinding algorithm developed by Genentech, Inc. (South San Francisco,Calif.) upon ESTs as well as clustered and assembled EST fragments frompublic (e.g., Genbank) and/or private (LIFESEQ®, Incyte Pharmaceuticals,Inc., Palo Alto, Calif.) databases. The signal sequence algorithmcomputes a secretion signal score based on the character of the DNAnucleotides surrounding the first and optionally the second methioninecodon(s) (ATG) at the 5′-end of the sequence or sequence fragment underconsideration. The nucleotides following the first ATG must code for atleast 35 unambiguous amino acids without any stop codons. If the firstATG has the required amino acids, the second is not examined. If neithermeets the requirement, the candidate sequence is not scored. In order todetermine whether the EST sequence contains an authentic signalsequence, the DNA and corresponding amino acid sequences surrounding theATG codon are scored using a set of seven sensors (evaluationparameters) known to be associated with secretion signals.

Use of the above described signal sequence algorithm allowedidentification of an EST cluster sequence from the LIFESEQ® database,Incyte Pharmaceuticals, Palo Alto, Calif., designated herein asCLU98611. This EST cluster sequence was then compared to a variety ofexpressed sequence tag (EST) databases which included public ESTdatabases (e.g., Genbank) and a proprietary EST DNA database (LIFESEQ®,Incyte Pharmaceuticals, Palo Alto, Calif.) to identify existinghomologies. The homology search was performed using the computer programBLAST or BLAST2 (Altshul et al., Methods in Enzymology 266:460-480(1996)). Those comparisons resulting in a BLAST score of 70 (or in somecases 90) or greater that did not encode known proteins were clusteredand assembled into a consensus DNA sequence with the program “phrap”(Phil Green, University of Washington, Seattle, Wash.). The consensussequence obtained therefrom is herein designated DNA82392.

In light of an observed sequence homology between the DNA82392 sequenceand an EST sequence encompassed within clone no. 653153 from theLIFESEQ® database, Incyte Pharmaceuticals, Palo Alto, Calif., clone no.653153 was purchased and the cDNA insert was obtained and sequenced. Itwas found herein that that cDNA insert encoded a full-length protein.The sequence of this cDNA insert is shown in FIG. 79 and is hereindesignated as DNA96860-2700.

Clone DNA96860-2700 contains a single open reading frame with anapparent translational initiation site at nucleotide positions 83-85 andending at the stop codon at nucleotide positions 1667-1669 (FIG. 79; SEQID NO:79). The predicted polypeptide precursor is 528 amino acids long(FIG. 80). The full-length PRO6017 protein shown in FIG. 80 has anestimated molecular weight of about 59,000 daltons and a pI of about8.73. Analysis of the full-length PRO6017 sequence shown in FIG. 80 (SEQID NO: 80) evidences the presence of a variety of important polypeptidedomains as shown in FIG. 80, wherein the locations given for thoseimportant polypeptide domains are approximate as described above. CloneDNA96860-2700 has been deposited with ATCC on Aug. 3, 1999 and isassigned ATCC Deposit No. PTA-478.

An analysis of the Dayhoff database (version 35.45 SwissProt 35), usingthe ALIGN-2 sequence alignment analysis of the full-length sequenceshown in FIG. 80 (SEQ ID NO: 80), evidenced sequence identity betweenthe PRO6017 amino acid sequence and the following Dayhoff sequences:HSA011001_(—)1; P_W36903; HSHE6_(—)1; AF111092_(—)1; GEN14046; P_W48756;AC004262_(—)1; AF031573_(—)1; P_WO7600; P_W37412.

Example 41 Isolation of cDNA Clones Encoding Human PRO7174 Polypeptides[UNQ2784]

DNA96883-2745 was identified by applying a proprietary signal sequencefinding algorithm developed by Genentech, Inc. (South San Francisco,Calif.) upon ESTs as well as clustered and assembled EST fragments frompublic (e.g., Genbank) and/or private (LIFESEQ®, Incyte Pharmaceuticals,Inc., Palo Alto, Calif.) databases. The signal sequence algorithmcomputes a secretion signal score based on the character of the DNAnucleotides surrounding the first and optionally the second methioninecodon(s) (ATG) at the 5′-end of the sequence or sequence fragment underconsideration. The nucleotides following the first ATG must code for atleast 35 unambiguous amino acids without any stop codons. If the firstATG has the required amino acids, the second is not examined. If neithermeets the requirement, the candidate sequence is not scored. In order todetermine whether the EST sequence contains an authentic signalsequence, the DNA and corresponding amino acid sequences surrounding theATG codon are scored using a set of seven sensors (evaluationparameters) known to be associated with secretion signals.

Use of the above described signal sequence algorithm allowedidentification of an EST cluster sequence from the LIFESEQ® database,Incyte Pharmaceuticals, Palo Alto, Calif., designated herein asCLU92188. This EST cluster sequence was then compared to a variety ofexpressed sequence tag (EST) databases which included public ESTdatabases (e.g., Genbank) and a proprietary EST DNA database (LIFESEQ®,Incyte Pharmaceuticals, Palo Alto, Calif.) to identify existinghomologies. The homology search was performed using the computer programBLAST or BLAST2 (Altshul et al., Methods in Enzymology 266:460-480(1996)). Those comparisons resulting in a BLAST score of 70 (or in somecases 90) or greater that did not encode known proteins were clusteredand assembled into a consensus DNA sequence with the program “phrap”(Phil Green, University of Washington, Seattle, Wash.). The consensussequence obtained therefrom is herein designated DNA83604.

In light of an observed sequence homology between the DNA83604 sequenceand an EST sequence encompassed within clone no. 3362284 from theLIFESEQ® database, Incyte Pharmaceuticals, Palo Alto, Calif., clone no.3362284 was purchased and the cDNA insert was obtained and sequenced. Itwas found herein that cDNA insert encoded a full-length protein. Thesequence of this cDNA insert is shown in FIG. 81 and is hereindesignated as DNA96883-2745.

Clone DNA96883-2745 contains a single open reading frame with anapparent translational initiation site at nucleotide positions 3-5 andending at the stop codon at nucleotide positions 1,545-1,547 (FIG. 81;SEQ ID NO:81). The predicted polypeptide precursor is 514 amino acidslong (FIG. 82). The full-length PRO7174 protein shown in FIG. 82 has anestimated molecular weight of about 55,687 daltons and a pI of about8.78. Analysis of the full-length PRO7174 sequence shown in FIG. 82 (SEQID NO: 82) evidences the presence of a variety of important polypeptidedomains as shown in FIG. 82, wherein the locations given for thoseimportant polypeptide domains are approximate as described above. CloneDNA96883-2745 has been deposited with ATCC on Aug. 17, 1999 and isassigned ATCC Deposit No. PTA-544.

An analysis of the Dayhoff database (version 35.45 SwissProt 35), usingthe ALIGN-2 sequence alignment analysis of the full-length sequenceshown in FIG. 82 (SEQ ID NO: 82), evidenced sequence identity betweenthe PRO7174 amino acid sequence and the following Dayhoff sequences:RNU44129_(—)1; ER53_HUMAN; XLU44130_(—)1; P_W88699; VP36_CANFA; G01447;P_W67846; P_W67963; HSERGICP02_(—)1; and CCAD_CHICK.

Example 42 Isolation of cDNA Clones Encoding Human PRO9744 Polypeptides[UNQ3003]

A cDNA clone (DNA136110-2763) encoding a native human PRO9744polypeptide was identified using a CARD domain containing molecule,SOCA-1. More particularly, a cDNA fragment encoding the N-terminalportion of SOCA-1 was used to screen a human fetal kidney library.Several positive colonies were picked up, DNA were prepared andsequenced. DNA sequencing revealed that one of the cDNA clones containsa full length open reading frame that encodes a protein, homologous tothe human Rac protein, designated herein DNA136110-2763.

Clone DNA136110-2763 contains a single open reading frame with anapparent translational initiation site at nucleotide positions 242-244and ending at the stop codon at nucleotide positions 1334-1336 (FIG. 83;SEQ ID NO:83). The predicted polypeptide precursor is 364 amino acidslong (FIG. 84; SEQ ID NO:84). The full-length PRO9744 protein shown inFIG. 84 has an estimated molecular weight of about 42195 daltons and apI of about 7.4. Analysis of the full-length PRO9744 sequence shown inFIG. 84 (SEQ ID NO:84) evidences the presence of a variety of importantpolypeptide domains as shown in FIG. 84, wherein the locations given forthose important polypeptide domains are approximate as described above.Clone DNA136110-2763 has been deposited with ATCC on Sep. 14, 1999 andis assigned ATCC deposit no. PTA-652.

An analysis of the Dayhoff database (version 35.45 SwissProt 35), usingthe ALIGN-2 sequence alignment analysis of the full-length sequenceshown in FIG. 84 (SEQ ID NO: 84), evidenced sequence identity betweenthe PRO9744 amino acid sequence and the following Dayhoff sequences:KRAC_DICDI, KAPC_DICDI, PK2_DICDI, KAPC_DROME, GEN13181, GEN12288,P_R95911, TCU63742_(—)1, SGK_HUMAN, and AF135794_(—)1.

Example 43 Isolation of cDNA Clones Encoding Human PRO9821 Polypeptides[UNQ3023]

DNA108725-2766 was identified by applying a proprietary signal sequencefinding algorithm developed by Genentech, Inc. (South San Francisco,Calif.) upon ESTs as well as clustered and assembled EST fragments frompublic (e.g., GenBank) and/or private (LIFESEQ®, Incyte Pharmaceuticals,Inc., Palo Alto, Calif.) databases. The signal sequence algorithmcomputes a secretion signal score based on the character of the DNAnucleotides surrounding the first and optionally the second methioninecodon(s) (ATG) at the 5′-end of the sequence or sequence fragment underconsideration. The nucleotides following the first ATG must code for atleast 35 unambiguous amino acids without any stop codons. If the firstATG has the required amino acids, the second is not examined. If neithermeets the requirement, the candidate sequence is not scored. In order todetermine whether the EST sequence contains an authentic signalsequence, the DNA and corresponding amino acid sequences surrounding theATG codon are scored using a set of seven sensors (evaluationparameters) known to be associated with secretion signals.

Use of the above described signal sequence algorithm allowedidentification of an EST sequence from the Incyte database, designatedherein as DNA21277. This EST sequence was then compared to a variety ofexpressed sequence tag (EST) databases which included public ESTdatabases (e.g., GenBank) and a proprietary EST DNA database (LIFESEQ®,Incyte Pharmaceuticals, Palo Alto, Calif.) to identify existinghomologies. The homology search was performed using the computer programBLAST or BLAST2 (Altshul et al., Methods in Enzymology 266:460-480(1996)). Those comparisons resulting in a BLAST score of 70 (or in somecases 90) or greater that did not encode known proteins were clusteredand assembled into a consensus DNA sequence with the program “phrap”(Phil Green, University of Washington, Seattle, Wash.). The consensussequence obtained therefrom is herein designated DNA91971.

In light of an observed sequence homology between the DNA91971 sequenceand an EST sequence encompassed within clone no. 3232833H1 from theIncyte database, clone no. 3232833 was purchased and the cDNA insert wasobtained and sequenced. It was found herein that that cDNA insertencoded a full-length protein. The sequence of this cDNA insert is shownin FIG. 85 and is herein designated as DNA108725-2766.

Clone DNA108725-2766 contains a single open reading frame with anapparent translational initiation site at nucleotide positions 196-198and ending at the stop codon at nucleotide positions 709-711 (FIG. 85;SEQ ID NO: 85). The predicted polypeptide precursor is shown in FIG. 86.The full-length PRO9821 protein shown in FIG. 86 has an estimatedmolecular weight of about 19118 daltons and a pI of about 5.99. Analysisof the full-length PRO9821 sequence shown in FIG. 86 (SEQ ID NO:86)evidences the presence of a variety of important polypeptide domains asshown in FIG. 86, wherein the locations given for those importantpolypeptide domains are approximate as described above. CloneDNA108725-2766 has been deposited with ATCC on Oct. 19, 1999 and isassigned ATCC deposit no. PTA-863.

An analysis of the Dayhoff database (version 35.45 SwissProt 35), usingthe ALIGN-2 sequence alignment analysis of the full-length sequenceshown in FIG. 86 (SEQ ID NO: 86), evidenced sequence identity betweenthe PRO9821 amino acid sequence and the following Dayhoff sequences:P_Y27573; UPAR_MOUSE; UPAR_RAT; S42152; SP63_STRPU; AF007789_(—)1;CELR11F4_(—)1; LY6A_MOUSE; P_Y02738; and AF141377_(—)1.

Example 44 Isolation of cDNA Clones Encoding Human PRO9852 Polypeptides[UNQ3037]

DNA129332-2775 was identified by applying a proprietary signal sequencefinding algorithm developed by Genentech, Inc. (South San Francisco,Calif.) upon ESTs as well as clustered and assembled EST fragments frompublic (e.g., Genbank) and/or private (LIFESEQ®, Incyte Pharmaceuticals,Inc., Palo Alto, Calif.) databases. The signal sequence algorithmcomputes a secretion signal score based on the character of the DNAnucleotides surrounding the first and optionally the second methioninecodon(s) (ATG) at the 5′-end of the sequence or sequence fragment underconsideration. The nucleotides following the first ATG must code for atleast 35 unambiguous amino acids without any stop codons. If the firstATG has the required amino acids, the second is not examined. If neithermeets the requirement, the candidate sequence is not scored. In order todetermine whether the EST sequence contains an authentic signalsequence, the DNA and corresponding amino acid sequences surrounding theATG codon are scored using a set of seven sensors (evaluationparameters) known to be associated with secretion signals.

Use of the above described signal sequence algorithm allowedidentification of the DNA124065 consensus sequence and oligonucleotideswere synthesized based on this sequence. These oligonucleotides wereused 1) to identify by PCR a cDNA library that contained the sequence ofinterest, and 2) for use as probes to isolate a clone of the full-lengthcoding sequence for PRO9852. Forward and reverse PCR primers generallyrange from 20 to 30 nucleotides and are often designed to give a PCRproduct of about 100-1000 bp in length. The probe sequences aretypically 40-55 bp in length. In some cases, additional oligonucleotidesare synthesized when the consensus sequence is greater than about 1-1.5kbp. In order to screen several libraries for a full-length clone, DNAfrom the libraries was screened by PCR amplification, as per Ausubel etal., Current Protocols in Molecular Biology, supra, with the PCR primerpair. A positive library was then used to isolate clones encoding thegene of interest using the probe oligonucleotide and one of the primerpairs.

PCR primers (forward and reverse) were synthesized:

forward PCR primer 5′-CGATACTCGCGGGAGGCTAAC-3′ (SEQ ID NO: 174)reverse PCR primer 5′-CCTTCTGGGTGTCTCCAGTTAGCG-3′ (SEQ ID NO: 175)Additionally, a synthetic oligonucleotide hybridization probe wasconstructed from the consensus DNA124065 sequence which had thefollowing nucleotide sequence

hybridization probe (SEQ ID NO: 176)5′-CAACTCGCGCACTCAAAGATGGTCCCCATCCCTGCTG-3′

RNA for construction of the cDNA libraries was isolated from human FetalKidney tissue. The cDNA libraries used to isolate the cDNA clones wereconstructed by standard methods using commercially available reagentssuch as those from Invitrogen, San Diego, Calif. The cDNA was primedwith oligo dT containing a NotI site, linked with blunt to SalIhemikinased adaptors, cleaved with NotI, sized appropriately by gelelectrophoresis, and cloned in a defined orientation into a suitablecloning vector (such as pRKB or pRKD; pRK5B is a precursor of pRK5D thatdoes not contain the SfiI site; see, Holmes et al., Science,253:1278-1280 (1991)) in the unique XhoI and NotI sites.

DNA sequencing of the clones isolated as described above gave thefull-length DNA sequence for a full-length PRO9852 polypeptide(designated herein as DNA129332-2775 [FIG. 87, SEQ ID NO: 87]) and thederived protein sequence for that PRO9852 polypeptide.

Clone DNA129332-2775 contains a single open reading frame with anapparent translational initiation site at nucleotide positions 18-20 andending at the stop codon at nucleotide positions 1296-1298 (FIG. 87).The predicted polypeptide precursor is 426 amino acids long (FIG. 88).The full-length PRO9852 protein shown in FIG. 88 has an estimatedmolecular weight of about 46,884 daltons and a pI of about 7.01.Analysis of the full-length PRO9852 sequence shown in FIG. 88 (SEQ IDNO:88) evidences the presence of a variety of important polypeptidedomains as shown in FIG. 88, wherein the locations given for thoseimportant polypeptide domains are approximate as described above. CloneDNA129332-2775 has been deposited with ATCC on Nov. 9, 1999 and isassigned ATCC Deposit No. PTA-944.

An analysis of the Dayhoff database (version 35.45 SwissProt 35), usingthe ALIGN-2 sequence alignment analysis of the full-length sequenceshown in FIG. 88 (SEQ ID NO: 88), evidenced sequence identity betweenthe PRO9852 amino acid sequence and the following Dayhoff sequences:C70643; P_Y19557; A72114; B71551; S74705; H70793; F69812; T08715;P_Y34750; P_W14450.

Example 45 Isolation of cDNA Clones Encoding Human PRO9873 Polypeptides[UNQ3054]

The extracellular domain (ECD) sequences (including the secretion signalsequence, if any) from about 950 known secreted proteins from theSwiss-Prot public database were used to search EST databases. The ESTdatabases included a (1) public EST databases (e.g., GenBank), (2) aproprietary EST database (LIFESEQ®, Incyte Pharmaceuticals, Palo Alto,Calif.), and (3) a proprietary EST database from Genentech. The searchwas performed using the computer program BLAST or BLAST2 [Altschul etal., Methods in enzymology, 266:460-480 (1996)] as a comparison of theECD protein sequences to a 6 frame translation of the EST sequences.Those comparisons resulting in a BLAST score of 70 (or in some cases,90) or greater that did not encode known proteins were clustered andassembled into consensus DNA sequences with the program “phrap” (PhilGreen, University of Washington, Seattle, Wash.).

A consensus DNA sequence was assembled relative to other EST sequencesusing phrap as described above. This consensus sequence is hereindesignated DNA117942. In some cases, the consensus sequence derives froman intermediate consensus DNA sequence which was extended using repeatedcycles of BLAST and phrap to extend that intermediate consensus sequenceas far as possible using the sources of EST sequences discussed above.

Based on the DNA 117942 consensus sequence oligonucleotides weresynthesized: 1) to identify by PCR a cDNA library that contained thesequence of interest, and 2) for use as probes to isolate a clone of thefull-length coding sequence for PRO9873. Forward and reverse PCR primersgenerally range from 20 to 30 nucleotides and are often designed to givea PCR product of about 100-1000 bp in length. The probe sequences aretypically 40-55 bp in length. In some cases, additional oligonucleotidesare synthesized when the consensus sequence is greater than about 1-1.5kbp. In order to screen several libraries for a full-length clone, DNAfrom the libraries was screened by PCR amplification, as per Ausubel etal., Current Protocols in Molecular Biology, supra, with the PCR primerpair. A positive library was then used to isolate clones encoding thegene of interest using the probe oligonucleotide and one of the primerpairs.

PCR primers (forward and reverse) were synthesized:

forward PCR primer (SEQ ID NO: 177) 5′-CAGAAAAAAGGAAGATGGCAAG-3′;forward PCR primer (SEQ ID NO: 178) 5′-GGCAAGAATATTGTTACTTTTCCTCCCG-3′;reverse PCR primer (SEQ ID NO: 179) 5′-TTACCAGCTTTGAGTACACATAGA-3′; andreverse PCR primer (SEQ ID NO: 180) 5′-AACGTTAATGAATCTACAGTCCGGGGC-3′.Additionally, a synthetic oligonucleotide hybridization probes wereconstructed from the consensus DNA 117942 sequence which had thefollowing nucleotide sequence

hybridization probe (SEQ ID NO: 181)5′-GGTCCATAAATATTCCATGCACAGCACATACAGCCACAAGACCCGGG AGG-3′; andhybridization probe (SEQ ID NO: 182)5′-TGTGCATGGAATATTTATGGACCGTCTAGCTTCCAAGAAG-3′;

RNA for construction of the cDNA libraries was isolated from human braintissue. The cDNA libraries used to isolate the cDNA clones wereconstructed by standard methods using commercially available reagentssuch as those from Invitrogen, San Diego, Calif. The cDNA was primedwith oligo dT containing a NotI site, linked with blunt to SalIhemikinased adaptors, cleaved with NotI, sized appropriately by gelelectrophoresis, and cloned in a defined orientation into a suitablecloning vector (such as pRKB or pRKD; pRK5B is a precursor of pRK5D thatdoes not contain the SfiI site; see, Holmes et al., Science,253:1278-1280 (1991)) in the unique XhoI and NotI sites.

DNA sequencing of the clones isolated as described above gave thefull-length DNA sequence for a full-length PRO9873 polypeptide(designated herein as DNA143076-2787 [FIG. 89, SEQ ID NO: 89]) and thederived protein sequence for that PRO9873 polypeptide.

The full length clone identified above contained a single open readingframe with an apparent translational initiation site at nucleotidepositions 38-40 and a stop signal at nucleotide positions 422-444 (FIG.89, SEQ ID NO:89). The predicted polypeptide precursor is 128 aminoacids long, has a calculated molecular weight of approximately 14332daltons and an estimated pI of approximately 4.83. Analysis of thefull-length PRO9873 sequence shown in FIG. 90 (SEQ ID NO:90) evidencesthe presence of a variety of important polypeptide domains as shown inFIG. 90, wherein the locations given for those important polypeptidedomains are approximate as described above. Clone DNA143076-2787 hasbeen deposited with ATCC on Dec. 7, 1999 and is assigned ATCC depositno. PTA-1028.

An analysis of the Dayhoff database (version 35.45 SwissProt 35), usingthe ALIGN-2 sequence alignment analysis of the full-length sequenceshown in FIG. 90 (SEQ ID NO:90), evidenced sequence identity between thePRO9873 amino acid sequence and the following Dayhoff sequences:MIA_HUMAN, A42965_(—)1, P_R69811, MIA_BOVIN, RNU67884_(—)1, GEN14164,MIA_MOUSE, P_R69812, P_Y24788, and P_Y22236.

Example 46 Isolation of cDNA Clones Encoding Human PRO10196 Polypeptides[UNQ3115]

The extracellular domain (ECD) sequences (including the secretion signalsequence, if any) from about 950 known secreted proteins from theSwiss-Prot public database were used to search EST databases. The ESTdatabases included (1) public EST databases (e.g., GenBank), and (2) aproprietary EST database (LIFESEQ®, Incyte Pharmaceuticals, Palo Alto,Calif.). The search was performed using the computer program BLAST orBLAST2 [Altschul et al., Methods in Enzymology, 266:460-480 (1996)] as acomparison of the ECD protein sequences to a 6 frame translation of theEST sequences. Those comparisons resulting in a BLAST score of 70 (or insome cases, 90) or greater that did not encode known proteins wereclustered and assembled into consensus DNA sequences with the program“phrap” (Phil Green, University of Washington, Seattle, Wash.).

A consensus DNA sequence was assembled relative to other EST sequencesusing phrap as described above. This consensus sequence is hereindesignated DNA139146. In some cases, the consensus sequence derives froman intermediate consensus DNA sequence which was extended using repeatedcycles of BLAST and phrap to extend that intermediate consensus sequenceas far as possible using the sources of EST sequences discussed above.

EST clone no. 5398353 was then purchased from LIFESEQ®, IncytePharmaceuticals, Palo Alto, Calif., and the cDNA insert of that clonewas obtained and sequenced in entirety.

DNA sequencing of the insert obtained from the above clone gave thefull-length DNA sequence for a full-length PRO10196 polypeptide(designated herein as DNA144841-2816 [FIG. 91, SEQ ID NO: 91]) and thederived protein sequence for that PRO10196 polypeptide.

The full length clone identified above contained a single open readingframe with an apparent translational initiation site at nucleotidepositions 151-153 and a stop signal at nucleotide positions 775-777(FIG. 91, SEQ ID NO:91). The predicted polypeptide precursor is 208amino acids long, has a calculated molecular weight of approximately22187 daltons and an estimated pI of approximately 5.08. Analysis of thefull-length PRO10196 sequence shown in FIG. 92 (SEQ ID NO:92) evidencesthe presence of a variety of important polypeptide domains as shown inFIG. 92, wherein the locations given for those important polypeptidedomains are approximate as described above. Clone DNA144841-2816 hasbeen deposited with ATCC on Jan. 11, 2000 and is assigned ATCC depositno. PTA-1188.

An analysis of the Dayhoff database (version 35.45 SwissProt 35), usingthe ALIGN-2 sequence alignment analysis of the full-length sequenceshown in FIG. 92 (SEQ ID NO:92), evidenced sequence identity between thePRO10196 amino acid sequence and the following Dayhoff sequences:P_Y08581, rFGF19_(—)1, AB018122_(—)1, AF110400_(—)1, P_Y08582,FGFF_MOUSE, FGF6_MOUSE, P_R80781 and P_R70825.

Example 47 Isolation of cDNA Clones Encoding Human PRO21956 Polypeptides[UNQ6973]

The extracellular domain (ECD) sequences (including the secretion signalsequence, if any) from about 950 known secreted proteins from theSwiss-Prot public database were used to search sequence databases. Thedatabases included public databases (e.g., GenBank) In this instance,genomic DNA sequence from GenBank was analyzed using the gene predictionprogram GEN SCAN, licenced from Stanford University. GENSCAN analysispredicts gene coding regions, creating sequences which can be subjectedto the ECD search. The search was performed using the computer programBLAST or BLAST2 [Altschul et al., Methods in enzymology, 266:460-480(1996)] as a comparison of the ECD protein sequences to a 6 frametranslation of the sequences. Those comparisons resulting in a BLASTscore of 70 (or in some cases, 90) or greater that did not encode knownproteins were clustered and assembled into consensus DNA sequences withthe program “phrap” (Phil Green, University of Washington, Seattle,Wash.) if necessary.

A consensus DNA sequence was assembled. This consensus sequence isherein designated DNA146822.

Based on the DNA146822 consensus sequence, oligonucleotides weresynthesized: 1) to identify by PCR a cDNA library that contained thesequence of interest, and 2) for use as probes to isolate a clone of thefull-length coding sequence for PRO21956. Forward and reverse PCRprimers generally range from 20 to 30 nucleotides and are often designedto give a PCR product of about 100-1000 bp in length. The probesequences are typically 40-55 bp in length. In some cases, additionaloligonucleotides are synthesized when the consensus sequence is greaterthan about 1-1.5 kbp. In order to screen several libraries for afull-length clone, DNA from the libraries was screened by PCRamplification, as per Ausubel et al., Current Protocols in MolecularBiology, supra, with the PCR primer pair. A positive library was thenused to isolate clones encoding the gene of interest using the probeoligonucleotide and one of the primer pairs.

PCR primers (forward and reverse) were synthesized:

forward PCR primer 5′-ACAGCACCAAGTTTCTGAGCAACTTCCT-3′ (SEQ ID NO: 183)reverse PCR primer 5′-ACTTGAGGTTGTCACCGCACACG-3′ (SEQ ID NO: 184)Additionally, a synthetic oligonucleotide hybridization probe wasconstructed from the consensus DNA146822 sequence which had thefollowing nucleotide sequence

hybridization probe (SEQ ID NO: 185)5′-AGAGAGGAAACAAGGACCTGCGGGCACGGGCAGACG-3′

A pool of 50 different human cDNA libraries from various tissues wasused in cloning. The cDNA libraries used to isolate the cDNA clones wereconstructed by standard methods using commercially available reagentssuch as those from Invitrogen, San Diego, Calif. The cDNA was primedwith oligo dT containing a NotI site, linked with blunt to SalIhemikinased adaptors, cleaved with NotI, sized appropriately by gelelectrophoresis, and cloned in a defined orientation into a suitablecloning vector (such as pRKB or pRKD; pRK5B is a precursor of pRK5D thatdoes not contain the SfiI site; see, Holmes et al., Science,253:1278-1280 (1991)) in the unique XhoI and NotI sites.

DNA sequencing of the clones isolated as described above gave thefull-length DNA sequence for a full-length PRO21956 polypeptide(designated herein as DNA 178511-2986 [FIG. 97, SEQ ID NO: 97) and thederived protein sequence for that PRO21956 polypeptide.

The full length clone identified above contained a single open readingframe with an apparent translational initiation site at nucleotidepositions 74-76 and a stop signal at nucleotide positions 1145-1147(FIG. 97, SEQ ID NO:97). The predicted polypeptide precursor is 357amino acids long, has a calculated molecular weight of approximately39001 daltons and an estimated pI of approximately 9.28. Analysis of thefull-length PRO21956 sequence shown in FIG. 98 (SEQ ID NO:98) evidencesthe presence of a variety of important polypeptide domains as shown inFIG. 98, wherein the locations given for those important polypeptidedomains are approximate as described above. Clone DNA178511-2986 hasbeen deposited with ATCC on Sep. 12, 2000 and is assigned ATCC depositno. PTA-2452.

An analysis of the protein database (version 35.45 SwissProt 35), usingthe ALIGN-2 sequence alignment analysis of the full-length sequenceshown in FIG. 98 (SEQ ID NO:98), evidenced sequence identity between thePRO21956 amino acid sequence and the following protein sequences:WN14_CHICK.

Example 48

Generation and Analysis of Mice Comprising PRO226, PRO257, PRO268,PRO290, PRO36006, PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071,PRO1125, PRO1134, PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387,PRO1419, PRO1433, PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904,PRO35193, PRO4341, PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985,PRO4989, PRO5737, PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821,PRO9852, PRO9873, PRO10196, PRO34778, PRO20233, PRO21956, PRO57290,PRO38465, PRO38683 or PRO85161 Gene Disruptions

To investigate the role of PRO226, PRO257, PRO268, PRO290, PRO36006,PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125, PRO1134,PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433,PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341,PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737,PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873,PRO10196, PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 orPRO85161 polypeptides, disruptions in PRO226, PRO257, PRO268, PRO290,PRO36006, PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125,PRO1134, PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419,PRO1433, PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193,PRO4341, PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989,PRO5737, PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852,PRO9873, PRO10196, PRO34778, PRO20233, PRO21956, PRO57290, PRO38465,PRO38683 or PRO85161 genes were produced by homologous recombination orretroviral insertion techniques. Specifically, transgenic micecomprising disruptions in PRO226, PRO257, PRO268, PRO290, PRO36006,PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125, PRO1134,PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433,PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341,PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737,PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873,PRO10196, PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 orPRO85161 genes (i.e., knockout mice) were created by either genetargeting or gene trapping. Mutations were confirmed by southern blotanalysis to confirm correct targeting on both the 5′ and 3′ ends.Gene-specific genotyping was also performed by genomic PCR to confirmthe loss of the endogenous native transcript as demonstrated by RT-PCRusing primers that anneal to exons flanking the site of insertion.Targeting vectors were electroporated into 129 strain ES cells andtargeted clones were identified. Targeted clones were microinjected intohost blastocysts to produce chimeras. Chimeras were bred with C57animals to produce F1 heterozygotes. Heterozygotes were intercrossed toproduce F2 wild-type, heterozygote and homozygote cohorts which wereused for phenotypic analysis. Rarely, if not enough F1 heterozygoteswere produced, the F1 hets were bred to wild-type C57 mice to producesufficient heterozygotes to breed for cohorts to be analyzed for aphenotype. All phenotypic analysis was performed from 12-16 weeks afterbirth.

Overall Summary of Phenotypic Results

48.1. Generation and Analysis of Mice Comprising DNA33460-1166 (UNQ200)Gene Disruptions

In these knockout experiments, the gene encoding PRO226 polypeptides(designated as DNA33460-1166) (UNQ200) was disrupted. The gene specificinformation for these studies is as follows: the mutated mouse genecorresponds to nucleotide reference: NM_(—)021474 Mus musculus epidermalgrowth factor-containing fibulin-like extracellular matrix protein 2(Efemp2); protein reference: Q9JM06 Q9JM06 Q9JM06 EGF-CONTAININGFIBULIN-LIKE EXTRACELLULAR M; the human gene sequence reference:NM_(—)016938 ACCESSION: NM_(—)016938 NID: 8393298 Homo sapiensEGF-containing fibulin-like extracellular matrix protein 2 (EFEMP2); thehuman protein sequence corresponds to reference: 095967 FBL4_HUMAN095967 EGF-CONTAINING FIBULIN-LIKE EXTRACELLUL.

The mouse gene of interest is Efemp2 (epidermal growth factor-containingfibulin-like extracellular matrix protein 2), ortholog of human EFEMP2.Aliases include MBP1, UPH1, FBLN4, 0610011K11Rik, fibulin 4, andfibulin-4.

EFEMP2 is a secreted protein that likely functions as an extracellularmatrix protein. The protein contains a signal peptide, six epidermalgrowth factor (EGF)-like domains, and a globular fibulin-type module.EFEMP2 is prominently expressed in the medial layers of large veins andarteries as well as in a wide variety of other tissues. EFEMP2 may playa role in processes such as blood coagulation, complement activation,and cell fate determination during development. EFEMP2 is a candidategene for retinopathies that map to chromosome 11 (Katsanis et al, HumGenet 106(1):66-72 (2000); Argraves et al, EMBO Rep 4(12): 1127-31(2003)).

Targeted or gene trap mutations are generated in strain129SvEv^(Brd)-derived embryonic stem (ES) cells. The chimeric mice arebred to C57BL/6J albino mice to generate F1 heterozygous animals. Theseprogeny are intercrossed to generate F2 wild type, heterozygous, andhomozygous mutant progeny. On rare occasions, for example when very fewF1 mice are obtained from the chimera, F1 heterozygous mice are crossedto 129SvEv^(Brd)/C57 hybrid mice to yield additional heterozygousanimals for the intercross to generate the F2 mice. Level I phenotypicanalysis is performed on mice from this generation

wt het hom Total Observed 15 43 0 58 Expected 14.5 29 14.5 58 Chi-Sq. =20.29 Significance = 3.9271934E−5 (hom/n) = 0.09 Avg. Litter Size = 8Mutation InformationMutation Type Homologous Recombination (standard)Description: Coding exons 1 through 3 were targeted (NCBI accessionNM_(—)021474.1).1. Wild-type Expression Panel: Expression of the target gene wasdetected in embryonic stem (ES) cells and in all 13 adult tissue samplestested by RT-PCR.2. QC Expression: Disruption of the target gene was confirmed bySouthern hybridization analysis.

48.1.1. Phenotypic Analysis (for Disrupted Gene: DNA33460-1166 (UNQ200)

(a) Overall Phenotypic Summary:

Mutation of the gene encoding the ortholog of human epidermal growthfactor-containing fibulin-like extracellular matrix protein 2 (EFEMP2)resulted in late embryonic lethality of (−/−) mutants. Gene disruptionwas confirmed by Southern blot.

(b) Pathology

Microscopic: Embryonic lethal. At 12.5 days there were 49 embryosobserved: 12 (−/−) embryos, 19 (+/−) embryos, 9 (+/+) embryos, 8inconclusive and 1 inc-het-hom. No structural developmentalabnormalities were detected in these 12.5 d embryos by gross orhistological examination.

Discussion Related to Embryonic Developmental Abnormality of Lethality:

Embryonic lethality in knockout mice usually results from variousserious developmental problems including but not limited toneuro-degenerative diseases, angiogenic disorders, inflammatorydiseases, or where the gene/protein has an important role in basic cellsignaling processes in many cell types. In addition, embryonic lethalsare useful as potential cancer models. Likewise, the correspondingheterozygous (+/−) mutant animals are particularly useful when theyexhibit a phenotype and/or a pathology report which reveals highlyinformative clues as to the function of the knocked-out gene. Forinstance, EPO knockout animals were embryonic lethals, but the pathologyreports on the embryos showed a profound lack of RBCs.

48.2. Generation and Analysis of Mice Comprising DNA35841-1173 (UNQ224)Gene Disruptions

In these knockout experiments, the gene encoding PRO257 polypeptides(designated as DNA35841-1173) (UNQ224) was disrupted. The gene specificinformation for these studies is as follows: the mutated mouse genecorresponds to nucleotide reference: NM_(—)008411 ACCESSION:NM_(—)008411 NID: 11993940 Mus musculus Mus musculus integralmembrane-associated protein 1 (Itmap1); protein reference: P70412ACCESSION:P70412 NID: Mus musculus (Mouse). INTEGRAL MEMBRANE-ASSOCIATEDPROTEIN 1; the human gene sequence reference: NM_(—)022034 Homo sapiensCUB and zona pellucida-like domains 1 (CUZD1); the human proteinsequence corresponds to reference: Q86UP6 ACCESSION: Q86UP6 NID: Homosapiens (Human). Transmembrane protein UO-44D.

The mouse gene of interest is Cuzd1 (CUB and zona pellucida-like domains1), ortholog of human CUZD1. Aliases include USG, ERG-1, UO-44, UTCZP,Itmap1, integral membrane-associated protein 1, and estrogen regulatedgene 1.

CUZD1 is an integral membrane protein, containing a signal peptide, twoCUB (complement subcomponents C1r/C1s, sea urchin Uegf protein, bonemorphogenetic protein-1) domains, a zona pellucida (ZP) domain, and atransmembrane segment near the C-terminus (Kasik et al, Biochem J 330Pt2):947-50 (1998); Chen et al, J Biol Chem 275(7):5248 (1999)). CUB(Inter Pro accession IPR000859) and ZP (Pfam accession PF00100) domainsare generally found in extracellular proteins involved inprotein-protein interactions. The precise function of CUZD1 is notclear. CUZD1 is expressed in epithelia of normal ovarian tissue andovarian tumors and in epithelia from endometrium of pregnant uterus andoviduct, where CUZD1 is upregulated by estrogen and down-regulated byprogesterone (Chen et al, J Biol Chem 275(7):5248 (1999); Huynh et al,Endocrinology 142(7):2985-95 (2001)). In these epithelia, CUZD1 may belocated on the plasma membrane (Huynh et al, Endocrinology142(7):2985-95 (2001); Leong et al, Oncogene 23(33):5707-18 (2004) or ingranular structures in the apical region of uterine epithelium (Imamurae al, J Biol Chem 277(52):50725-33 (2002)). CUZD1 is also expressed onthe membrane of trypsinogen-containing zymogen granules of pancreaticacinar cells (Imamura et al, J Biol Chem 277(52):50725-33 (2002)). CUZD1may play a role in the reproductive cycle and pregnancy (Kasik, BiochemJ 330Pt 2):947-50 (1998); Chen et al, J Biol Chem 275(7):5248 (1999), incell motility and cell-cell interactions (Leong et al, Oncogene23(33):5707-18 (2004), in epithelial cell proliferation anddifferentiation (Huynh et al, Endocrinology 142(7):2985-95 (2001), andin digestion (Imamura e al, J Biol Chem 277(52):50725-33 (2002)).

Imamura and coworkers (J Biol Chem 277(52):50725-33 2002)) investigatedthe physiological role of CUZD1 using knockout mice. They showed thatsecretagogue- and diet-induced pancreatitis susceptibility was muchhigher in CUZD1 homozygous null mice than in wild-type mice.Reproduction did not seem to be affected. Imamura and coworkers proposedthat CUZD1 plays a role in modulating trypsinogen activation within thezymogen granule and that altered trypsinogen activation is associatedwith severity of pancreatitis.

Targeted or gene trap mutations are generated in strain129SvEv^(Brd)-derived embryonic stem (ES) cells. The chimeric mice arebred to C57BL/6J albino mice to generate F1 heterozygous animals. Theseprogeny are intercrossed to generate F2 wild type, heterozygous, andhomozygous mutant progeny. On rare occasions, for example when very fewF1 mice are obtained from the chimera, F1 heterozygous mice are crossedto 129SvEv^(Brd)/C57 hybrid mice to yield additional heterozygousanimals for the intercross to generate the F2 mice. Level I phenotypicanalysis is performed on mice from this generation

wt het hom Total Observed 26 38 17 81 Expected 20.25 40.5 20.25 81Chi-Sq. = 1.75 Significance = 0.416862 (hom/n) = 0.22 Avg. Litter Size =9Mutation InformationMutation Type: Homologous Recombination (standard)Description: Coding exon 1 was targeted (NCBI accession NM_(—)008411.2).1. Wild-type Expression Panel: Expression of the target gene wasdetected in brain; eye; spleen; kidney; skeletal muscle; stomach, smallintestine, and colon; heart; adipose; banded heart; skin fibroblast;prostate; and MG 12 DPC among 26 adult tissue samples tested by RT-PCR.2. QC Expression: Disruption of the Target Gene was Confirmed bySouthern Hybridization Analysis.

48.2.1. Phenotypic Analysis (for Disrupted Gene: DNA35841-1173 (UNQ224)

(a) Overall Phenotypic Summary:

Mutation of the gene encoding the ortholog of human CUB and zonapellucida-like domains 1 (CUZD1) resulted in increased serum IgG3 levelsin (−/−) mice as well as increased percentages of subsets of B cells.Gene disruption was confirmed by Southern blot.

(b) Immunology Phenotypic Analysis

Immune related and inflammatory diseases are the manifestation orconsequence of fairly complex, often multiple interconnected biologicalpathways which in normal physiology are critical to respond to insult orinjury, initiate repair from insult or injury, and mount innate andacquired defense against foreign organisms. Disease or pathology occurswhen these normal physiological pathways cause additional insult orinjury either as directly related to the intensity of the response, as aconsequence of abnormal regulation or excessive stimulation, as areaction to self, or as a combination of these.

Though the genesis of these diseases often involves multistep pathwaysand often multiple different biological systems/pathways, interventionat critical points in one or more of these pathways can have anameliorative or therapeutic effect. Therapeutic intervention can occurby either antagonism of a detrimental process/pathway or stimulation ofa beneficial process/pathway.

T lymphocytes (T cells) are an important component of a mammalian immuneresponse. T cells recognize antigens which are associated with aself-molecule encoded by genes within the major histocompatibilitycomplex (MHC). The antigen may be displayed together with MHC moleculeson the surface of antigen presenting cells, virus infected cells, cancercells, grafts, etc. The T cell system eliminates these altered cellswhich pose a health threat to the host mammal. T cells include helper Tcells and cytotoxic T cells. Helper T cells proliferate extensivelyfollowing recognition of an antigen-MHC complex on an antigen presentingcell. Helper T cells also secrete a variety of cytokines, i.e.,lymphokines, which play a central role in the activation of B cells,cytotoxic T cells and a variety of other cells which participate in theimmune response.

In many immune responses, inflammatory cells infiltrate the site ofinjury or infection. The migrating cells may be neutrophilic,eosinophilic, monocytic or lymphocytic as can be determined byhistological examination of the affected tissues. Current Protocols inImmunology, ed. John E. Coligan, 1994, John Wiley & Sons, Inc.

Many immune related diseases are known and have been extensivelystudied. Such diseases include immune-mediated inflammatory diseases(such as rheumatoid arthritis, immune mediated renal disease,hepatobiliary diseases, inflammatory bowel disease (IBD), psoriasis, andasthma), non-immune-mediated inflammatory diseases, infectious diseases,immunodeficiency diseases, neoplasia, and graft rejection, etc. In thearea of immunology, targets were identified for the treatment ofinflammation and inflammatory disorders.

In the area of immunology, targets have been identified herein for thetreatment of inflammation and inflammatory disorders. Immune relateddiseases, in one instance, could be treated by suppressing the immuneresponse. Using neutralizing antibodies that inhibit molecules havingimmune stimulatory activity would be beneficial in the treatment ofimmune-mediated and inflammatory diseases. Molecules which inhibit theimmune response can be utilized (proteins directly or via the use ofantibody agonists) to inhibit the immune response and thus ameliorateimmune related disease.

The following tests were performed:

Serum Immunoglobulin Isotyping Assay:

The Serum Immunoglobulin Isotyping Assay is performed using a CytometricBead Array (CBA) kit. This assay is used to rapidly identify the heavyand light chain isotypes of a mouse monoclonal antibody in a singlesample. The values expressed are “relative fluorescence units” and arebased on the detection of kappa light chains. Any value <6 is notsignificant.

Results:

Serum Imm. 2: The (−/−) mice exhibited an increased mean serum IgG3level when compared with that of their (+/+) littermates, the median forthe (+/+) mice, and the cumulative (+/+) historical median.

Mutant (−/−) mice exhibited increased IgG3 serum immunoglobulinscompared to their gender-matched (+/+) littermates. IgG3 immunoglobulinshave neutralization effects and to a lesser extent are important foractivation of the complement system. The observed phenotype suggeststhat the PRO257 polypeptide is a negative regulator of inflammatoryresponses. These immunological abnormalities suggest that antagonists orinhibitors of PRO257 polypeptides would be important agents which wouldstimulate the immune system (such as T cell proliferation) and wouldfind utility in the cases wherein this effect would be beneficial to theindividual such as in the case of leukemia, and other types of cancer,and in immuno-compromised patients, such as AIDS sufferers. Accordingly,PRO257 polypeptides would be useful in inhibiting the immune responseand would be useful candidates for suppressing harmful immune responses,e.g. in the case of graft rejection or graft-versus-host diseases.

Fluorescence-Activated Cell-Sorting (FACS) Analysis

Procedure:

FACS analysis of immune cell composition from peripheral blood wasperformed including CD4, CD8 and T cell receptor to evaluate Tlymphocytes, CD19 for B lymphocytes, CD45 as a leukocyte marker and panNK for natural killer cells. The FACS analysis was carried out on 2 wildtype and 6 homozygous mice and included cells derived from thymus,spleen, bone marrow and lymph node.

In these studies, analyzed cells were isolated from thymus, peripheralblood, spleen, bone marrow and lymph nodes. Flow cytometry was designedto determine the relative proportions of CD4 and CD8 positive T cells, Bcells, NK cells and monocytes in the mononuclear cell population. ABecton-Dickinson FACSCalibur 3-laser FACS machine was used to assessimmune status. For Phenotypic Assays and Screening, this machine recordsCD4+/CD8−, CD8+/CD4−, NK, B cell and monocyte numbers in addition to theCD4+/CD8+ ratio.

The mononuclear cell profile was derived by staining a single sample oflysed peripheral blood from each mouse with a panel of sixlineage-specific antibodies: CD45 PerCP, anti-TCRb APC, CD4 PE, CD8FITC, pan-NK PE, and CD19 FITC. The two FITC and PE labeled antibodiesstain mutually exclusive cell types. The samples were analyzed using aBecton Dickinson FACSCalibur flow cytometer with CellQuest software.

Results:

Tissue Specific FACS-Project: The (−/−) mice exhibited an increasedpercentage of B220+ CD11b Low CD23−cells and decreased percentage ofB220+ CD11b− CD23+ cells in peritoneal lavage when compared with thoseof their (+/+) littermates.

48.3. Generation and Analysis of Mice Comprising DNA39427-1179 (UNQ235)Gene Disruptions

In these knockout experiments, the gene encoding PRO268 polypeptides(designated as DNA39427-1179) (UNQ235) was disrupted. The gene specificinformation for these studies is as follows: the mutated mouse genecorresponds to nucleotide reference: BC017603 ACCESSION:BC017603 NID:17160856 Mus musculus, RIKEN cDNA 2810425A04 gene, clone MGC:27603IMAGE:4503129; protein reference: Q8VBT0 ACCESSION: Q8VBT0 NID: Musmusculus (Mouse). RIKEN cDNA 2810425A04 GENE; the human gene sequencereference: NM_(—)030755 ACCESSION: NM_(—)030755 NID:13559515 Homosapiens thioredoxin domain-containing (TXNDC); the human proteinsequence corresponds to reference: Q9Y4T6 ACCESSION: Q9Y4T6 NID: Homosapiens (Human). HYPOTHETICAL 32.5 KDA PROTEIN (FRAGMENT).

The mouse gene of interest is Txndc1 (thioredoxin domain containing 1),ortholog of human TXNDC (thioredoxin domain containing). Aliases include2810425A04Rik, TMX, TXNDC1, DKFZP564E1962, thioredoxindomain-containing, and thioredoxin-related transmembrane protein.

TXNDC is an integral membrane protein located primarily in theendoplasmic reticulum that likely functions as a protein disulfideisomerase. The protein contains a signal peptide, a thioredoxin domain,and one or possibly two transmembrane segments. The thioredoxin domaincontains a dithiol active center that is capable of participatingreversibly in a variety of oxidation-reduction reactions. Moreover, thethioredoxin domain projects into the lumen of the endoplasmic reticulum,where it is likely to participate with other enzymes in protein foldingas well as in regulating redox state. Expression of TXNDC is ubiquitousbut is particularly high in lung, kidney, liver, and placenta (Matsuo etal, Arch Biochem Biophys 423(1):81-7 (2004); Matsuo et al, J Biol Chem276(13):10032-8 (2001)).

Targeted or gene trap mutations are generated in strain129SvEv^(Brd)-derived embryonic stem (ES) cells. The chimeric mice arebred to C57BL/6J albino mice to generate F1 heterozygous animals. Theseprogeny are intercrossed to generate F2 wild type, heterozygous, andhomozygous mutant progeny. On rare occasions, for example when very fewF1 mice are obtained from the chimera, F1 heterozygous mice are crossedto 129SvEv^(Brd)/C57 hybrid mice to yield additional heterozygousanimals for the intercross to generate the F2 mice. Level I phenotypicanalysis is performed on mice from this generation

wt het hom Total Observed 11 23 13 47 Expected 11.75 23.5 11.75 47Chi-Sq. = 7.33 Significance = 0.025604172 (hom/n) = 0.27 Avg. LitterSize = 10Mutation InformationMutation Type: Homologous Recombination (standard)Description: Coding exon 1 was targeted (NCBI accession NM_(—)028339.1).1. Wild-type Expression Panel: Expression of the target gene wasdetected in embryonic stem (ES) cells and in all 13 adult tissue samplestested by RT-PCR.2. QC Expression: Disruption of the target gene was confirmed bySouthern hybridization analysis.

48.3.1. Phenotypic Analysis (for Disrupted Gene: DNA39427-1179 (UNQ235)

(a) Overall Phenotypic Summary:

Mutation of the gene encoding the ortholog of human thioredoxin domaincontaining (TXNDC) resulted in decreased bone mineral densitymeasurements in the mutant (−/−) mice. Gene disruption was confirmed bySouthern blot.

(b) Bone Metabolism & Body Diagnostics: Radiology Phenotypic Analysis

In the area of bone metabolism, targets were identified herein for thetreatment of arthritis, osteoporosis, osteopenia and osteopetrosis aswell as identifying targets that promote bone healing. Tests included:

DEXA for measurement of bone mineral density on femur and vertebra

MicroCT for very high resolution and very high sensitivity measurementsof bone mineral density for both trabecular and cortical bone.

Dexa Analysis—Test Description:

Procedure: A cohort of 4 wild type, 4 heterozygous and 8 homozygous micewere tested in this assay. Dual Energy X-ray Absorptiometry (DEXA) hasbeen used successfully to identify changes in bone. Anesthetized animalswere examined and bone mineral content (BMC), BMC/LBM ratios, volumetricbone mineral density (vBMD), total body BMD, femur BMD and vertebra BMDwere measured.

The mouse was anesthetized by intraperitoneal injection of Avertin(1.25% 2,2,2,-tribromoethanol, 20 ml/kg body weight), body length andweight were measured, and then the mouse was placed in a prone positionon the platform of the PIXImus™ Densitometer (Lunar Inc.) for a DEXAscan. Using Lunar PIXImus software, the bone mineral density (BMD) andfat composition (% fat) and total tissue mass (TTM) were determined inthe regions of interest (ROI) [i.e., whole body, vertebrae, and bothfemurs].

Bone MicroCT Analysis:

Procedure: MicroCT was also used to get very sensitive measurements ofBMD. One vertebra and 1 femur were taken from a cohort of 4 wild typeand 8 homozygous mice. Measurements were taken of lumbar 5 vertebratrabecular bone volume, trabecular thickness, connectivity density andmidshaft femur total bone area and cortical thickness. The μCT40 scansprovided detailed information on bone mass and architecture. Multiplebones were placed into sample holders and scanned automatically.Instrument software was used to select regions of interest for analysis.Trabecular bone parameters were analyzed in the fifth lumbar vertebrae(LV5) at 16 micrometer resolution and cortical bone parameters wereanalyzed in the femur midshaft at a resolution of 20 micrometers.

Results:

DEXA: The male (−/−) mice exhibited decreased mean volumetric bonemineral density and bone mineral density in total body, femur, andvertebrae when compared with the levels for their gender-matched (+/+)littermates and the historical means.

Micro CT: The male (−/−) mice showed decreased mean femoral mid-shaftcortical thickness when compared with that of their gender-matched (+/+)littermates and the historical mean.

The (−/−) mice analyzed by DEXA and bone micro CT analysis exhibiteddecreased bone measurements when compared with their (+/+) littermates,suggestive of abnormal bone disorders. The negative bone phenotypeindicates that PRO268 polypeptides or agonists thereof would be usefulfor maintaining bone homeostasis. In addition, PRO268 polypeptides wouldbe useful in bone healing or for the treatment of arthritis orosteoporosis, whereas antagonists (or inhibitors) of PRO268 polypeptidesor its encoding gene would lead to abnormal or pathological bonedisorders including inflammatory diseases associated with abnormal bonemetabolism including arthritis, osteoporosis and osteopenia.

48.4. Generation and Analysis of Mice Comprising DNA35680-1212 (UNQ253)Gene Disruptions

In these knockout experiments, the gene encoding PRO290 polypeptides(designated as DNA35680-1212) (UNQ253) was disrupted. The gene specificinformation for these studies is as follows: the mutated mouse genecorresponds to nucleotide reference: XM_(—)150243 PREDICTED: Musmusculus cDNA sequence BC042396 (BC042396); protein reference:XP_(—)150243 mKIAA0540 protein [Mus musculus]; the human gene sequencereference: XM_(—)291064 PREDICTED: Homo sapiens KIAA0540 protein(KIAA0540); the human protein sequence corresponds to reference:XP_(—)291064 PREDICTED: KIAA0540 protein [Homo sapiens].

The mouse gene of interest is cDNA sequence BC042396, ortholog of humanKIAA0540 protein. Aliases include mKIAA0540 and 1110014F23Rik.

KIAA0540 protein contains a putative signal peptide or signal anchor, aBEACH domain (PFAM accession PF02138), and four tandem WD40 repeats(SMART accession SM00320). Bioinformatic analyses suggest that theprotein may be extracellular (Clark et al, Genome Res 13(10):2265-70(2003)). The domain organization of KIAA0540 protein is similar to thatof LYST (lysosomal trafficking regulator), which may be involved inprotein sorting to and from lysosomes and endosomes (Barbosa et al,Nature 385(6611):97 (1996)).

Targeted or gene trap mutations are generated in strain129SvEv^(Brd)-derived embryonic stem (ES) cells. The chimeric mice arebred to C57BL/6J albino mice to generate F1 heterozygous animals. Theseprogeny are intercrossed to generate F2 wild type, heterozygous, andhomozygous mutant progeny. On rare occasions, for example when very fewF1 mice are obtained from the chimera, F1 heterozygous mice are crossedto 129SvEv^(Brd)/C57 hybrid mice to yield additional heterozygousanimals for the intercross to generate the F2 mice. Level I phenotypicanalysis is performed on mice from this generation

wt het hom Total Observed 19 40 9 68 Expected 17.0 34.0 17.0 68 Chi-Sq.= 6.07 Significance = 0.04807466 (hom/n) = 0.19 Avg. Litter Size = 8Mutation InformationMutation Type: Homologous Recombination (standard)Description: Coding exons 4 through 11 were targeted (NCBI accessionXM_(—)150243.4).1. Wild-type Expression Panel: Expression of the target gene wasdetected in embryonic stem (ES) cells and in all 26 adult tissue samplestested by RT-PCR, except bone.2. QC Expression: Disruption of the target gene was confirmed bySouthern hybridization analysis.

48.4.1. Phenotypic Analysis (for Disrupted Gene: DNA35680-1212 (UNQ253)

(a) Overall Phenotypic Summary:

The homozygous mutant mice exhibited numerous immunologicalabnormalities, including an increased percentage of granulocytes, anincreased serum IL-6 response to LPS challenge, a decreased serum IgG2alevel, increase mean serum IgM levels; decreased mean percentages of CD4and CD8 cells in peripheral blood; and decreased T cell to B cell ratioin the spleen. In addition, the mutants exhibited neutrophils lackingthe granulation normally present in these cells. Decreased neutrophilswere also observed resulting in neutropenia. The (−/−) mice exhibiteddecreased platelets and increased platelet volume. Blood chemistryanalysis resulted in the observation of decreased mean serum glucoselevels and decreased mean serum triglycerides. The homozygous mutantmice also exhibited decreased bone measurements when compared with thoseof their gender-matched wild-type littermates. Disruption of the targetgene was confirmed by Southern hybridization analysis.

(b) Immunology Phenotypic Analysis

Immune related and inflammatory diseases are the manifestation orconsequence of fairly complex, often multiple interconnected biologicalpathways which in normal physiology are critical to respond to insult orinjury, initiate repair from insult or injury, and mount innate andacquired defense against foreign organisms. Disease or pathology occurswhen these normal physiological pathways cause additional insult orinjury either as directly related to the intensity of the response, as aconsequence of abnormal regulation or excessive stimulation, as areaction to self, or as a combination of these.

Though the genesis of these diseases often involves multistep pathwaysand often multiple different biological systems/pathways, interventionat critical points in one or more of these pathways can have anameliorative or therapeutic effect. Therapeutic intervention can occurby either antagonism of a detrimental process/pathway or stimulation ofa beneficial process/pathway.

T lymphocytes (T cells) are an important component of a mammalian immuneresponse. T cells recognize antigens which are associated with aself-molecule encoded by genes within the major histocompatabilitycomplex (MHC). The antigen may be displayed together with MHC moleculeson the surface of antigen presenting cells, virus infected cells, cancercells, grafts, etc. The T cell system eliminates these altered cellswhich pose a health threat to the host mammal. T cells include helper Tcells and cytotoxic T cells. Helper T cells proliferate extensivelyfollowing recognition of an antigen-MHC complex on an antigen presentingcell. Helper T cells also secrete a variety of cytokines, i.e.,lymphokines, which play a central role in the activation of B cells,cytotoxic T cells and a variety of other cells which participate in theimmune response.

In many immune responses, inflammatory cells infiltrate the site ofinjury or infection. The migrating cells may be neutrophilic,eosinophilic, monocytic or lymphocytic as can be determined byhistological examination of the affected tissues. Current Protocols inImmunology, ed. John E. Coligan, 1994, John Wiley & Sons, Inc.

Many immune related diseases are known and have been extensivelystudied. Such diseases include immune-mediated inflammatory diseases(such as rheumatoid arthritis, immune mediated renal disease,hepatobiliary diseases, inflammatory bowel disease (IBD), psoriasis, andasthma), non-immune-mediated inflammatory diseases, infectious diseases,immunodeficiency diseases, neoplasia, and graft rejection, etc. In thearea of immunology, targets were identified for the treatment ofinflammation and inflammatory disorders.

In the area of immunology, targets have been identified herein for thetreatment of inflammation and inflammatory disorders. Immune relateddiseases, in one instance, could be treated by suppressing the immuneresponse. Using neutralizing antibodies that inhibit molecules havingimmune stimulatory activity would be beneficial in the treatment ofimmune-mediated and inflammatory diseases. Molecules which inhibit theimmune response can be utilized (proteins directly or via the use ofantibody agonists) to inhibit the immune response and thus ameliorateimmune related disease.

The following tests were performed:

Hematology Analysis:

Test Description: Blood tests are carried out by Abbott's Cell-Dyn3500R, an automated hematology analyzer. Some of its features include afive-part WBC differential. ‘Patient’ reports can cover over 22parameters in all.

Results:

Hematology: The (−/−) mice exhibited a decreased neutrophil count whencompared with that of their (+/+) littermates and the historical mean.However, the neutropenia observed in the (−/−) mice by automatedanalysis was not confirmed by manual differentials. The (−/−) mice havea normal distribution of white blood cells, but the neutrophils do notexhibit the granulation normally present within the cells. The (−/−)mice also exhibited a decreased mean platelet count and increased meanplatelet volume.

These results indicate that the mutant (−/−) mice exhibited anabnormality related to neutropenia with abnormal granulation within theneutrophils observed. Neutrophils are the chief phagocytic leukocytes ofthe blood. Therefore, the (−/−) mice have a compromised ability to fightinfections.

In addition, the mutant mice deficient in the DNA35680-1212 generesulted in a phenotype related to coagulation disorders. In thisregard, PRO290 polypeptides or agonists thereof would be useful intreating disorders related to abnormal blood coagulation such ashemophilia.

Acute Phase Response:

Test Description: Bacterial lipopolysaccharide (LPS) is an endotoxin,and as such is a potent inducer of an acute phase response and systemicinflammation. The Level I LPS mice were injected intraperitoneally(i.p.) with a sublethal dose of LPS in 200 μL sterile saline using a 26gauge needle. The doses were based on the average weight of the micetested at 1 μg/g body weight 3 hours after injection; a 100 ul bloodsample was then taken and analyzed for the presence of TNFa, MCP-1, andIL-6 on the FACS Calibur instrument.

Results:

Acute Phase Response: The (−/−) mice exhibited an increased mean serumIL-6 response to LPS challenge when compared with that of their (+/+)littermates and the historical mean.

In summary, the LPS endotoxin challenge demonstrated that knockout micedeficient in the gene encoding PRO290 polypeptides exhibit immunologicalabnormalities when compared with their wild-type littermates. Inparticular, the mutant mice exhibited an increased ability to elicit animmunological response (IL-6 production) when challenged with the LPSendotoxin indicating a proinflammatory response. IL-6 contributes to thelater stages of B cell activation. In addition, IL-6 plays a criticalrole in inducing the acute phase response and systemic inflammation.This suggests that inhibitors or antagonists to PRO290 polypeptideswould stimulate the immune system and would find utility in the caseswherein this effect would be beneficial to the individual such as in thecase of leukemia, and other types of cancer, and in immuno-compromisedpatients, such as AIDS sufferers. Accordingly, PRO290 polypeptides oragonists thereof would be useful in inhibiting the immune response andwould be useful candidates for suppressing harmful immune responses,e.g. in the case of graft rejection or graft-versus-host diseases.

Serum Immunoglobulin Isotyping Assay:

The Serum Immunoglobulin Isotyping Assay is performed using a CytometricBead Array (CBA) kit. This assay is used to rapidly identify the heavyand light chain isotypes of a mouse monoclonal antibody in a singlesample. The values expressed are “relative fluorescence units” and arebased on the detection of kappa light chains. Any value <6 is notsignificant.

Results:

Serum Imm. 2: The (−/−) mice exhibited an increased mean serum IgM leveland a decreased mean serum IgG2a level when compared with those of their(+/+) littermates, the (+/+) mice within the project run, and thehistorical medians for each.

Mutant (−/−) mice exhibited elevation of IgM serum immunoglobulinscompared to their gender-matched (+/+) littermates. IgM immunoglobulinsare the first to be produced in a humoral immune response forneutralization of bacterial toxins and are particularly important inactivating the complement system. The observed phenotype suggests thatthe PRO290 polypeptide is a negative regulator of inflammatoryresponses. These immunological abnormalities suggest that inhibitors(antagonists) of PRO290 polypeptides would be useful in stimulating theimmune system (such as T cell proliferation) and would find utility inthe cases wherein this effect would be beneficial to the individual suchas in the case of leukemia, and other types of cancer, and inimmuno-compromised patients, such as AIDS sufferers. Accordingly, PRO290polypeptides or agonists thereof would be useful in inhibiting theimmune response and would be useful candidates for suppressing harmfulimmune responses, e.g. in the case of graft rejection orgraft-versus-host diseases.

The serum immunoglobulin isotyping assay also showed decreased orreduced levels of mean serum IgG2a in the homozygous (−/−) mice comparedto their gender-matched littermate (+/+) controls.

The serum immunoglobulin isotyping assay revealed that homozygous adultsexhibited decreased serum IgG2a levels. Thus, homozygotes showed anabnormally low serum immunoglobulins compared with the (+/+)littermates. Thus, the gene encoding PRO290 is essential for makingimmunoglobulins (or gamma globulins). Likewise, IgG2a immunoglobulinshave neutralization effects and to a lesser extent are important foractivation of the complement system.

Fluorescence-Activated Cell-Sorting (FACS) Analysis

Procedure:

FACS analysis of immune cell composition from peripheral blood wasperformed including CD4, CD8 and T cell receptor to evaluate Tlymphocytes, CD19 for B lymphocytes, CD45 as a leukocyte marker and panNK for natural killer cells. The FACS analysis was carried out on 2 wildtype and 6 homozygous mice and included cells derived from thymus,spleen, bone marrow and lymph node.

In these studies, analyzed cells were isolated from thymus, peripheralblood, spleen, bone marrow and lymph nodes. Flow cytometry was designedto determine the relative proportions of CD4 and CD8 positive T cells, Bcells, NK cells and monocytes in the mononuclear cell population. ABecton-Dickinson FACS Calibur 3-laser FACS machine was used to assessimmune status. For Phenotypic Assays and Screening, this machine recordsCD4+/CD8−, CD8+/CD4−, NK, B cell and monocyte numbers in addition to theCD4+/CD8+ ratio.

The mononuclear cell profile was derived by staining a single sample oflysed peripheral blood from each mouse with a panel of sixlineage-specific antibodies: CD45 PerCP, anti-TCRb APC, CD4 PE, CD8FITC, pan-NK PE, and CD19 FITC. The two FITC and PE labeled antibodiesstain mutually exclusive cell types. The samples were analyzed using aBecton Dickinson FACS Calibur flow cytometer with CellQuest software.

Results:

FACS3: The (−/−) mice exhibited an altered distribution of leukocytesubsets in the peripheral blood, characterized by decreased meanpercentages of CD4 and CD8 cells when compared with those of their (+/+)littermates and the historical means.

Tissue Specific FACS-Project: The (−/−) mice also exhibited an increasedpercentage of granulocytes by scatter but not an increased percentage ofB220− CD43 Hi cells in bone marrow when compared with that of their(+/+) littermates. The SSC-hi population was replaced by a large SSC-intpopulation in the (−/−) mice. The (−/−) mice also exhibited an increasedpercentage of B220 Hi CD23+ cells in peritoneal lavage.Tissue Specific FACS-Mouse: The (−/−) mice exhibited a decreased Tcell:B cell ratio and decreased percentages of CD62L Hi CD44 Dim CD4+and CD8+ cells in spleen when compared with that of their (+/+)littermates.

Thus, PRO290 polypeptides or agonists thereof act as a negativeregulator of B cell production.

(c) Bone Metabolism & Body Diagnostics: Radiology Phenotypic Analysis

In the area of bone metabolism, targets were identified herein for thetreatment of arthritis, osteoporosis, osteopenia and osteopetrosis aswell as identifying targets that promote bone healing. Tests included:

DEXA for measurement of bone mineral density on femur and vertebra

MicroCT for very high resolution and very high sensitivity measurementsof bone mineral density for both trabecular and cortical bone.

Dexa Analysis—Test Description:

Procedure: A cohort of 4 wild type, 4 heterozygous and 8 homozygous micewere tested in this assay. Dual Energy X-ray Absorptiometry (DEXA) hasbeen used successfully to identify changes in bone. Anesthetized animalswere examined and bone mineral content (BMC), BMC/LBM ratios, volumetricbone mineral density (vBMD), total body BMD, femur BMD and vertebra BMDwere measured.

The mouse was anesthetized by intraperitoneal injection of Avertin(1.25% 2,2,2,-tribromoethanol, 20 ml/kg body weight), body length andweight were measured, and then the mouse was placed in a prone positionon the platform of the PIXImus™ Densitometer (Lunar Inc.) for a DEXAscan. Using Lunar PIXImus software, the bone mineral density (BMD) andfat composition (% fat) and total tissue mass (TTM) were determined inthe regions of interest (ROI) [i.e., whole body, vertebrae, and bothfemurs].

Bone MicroCT Analysis:

Procedure: MicroCT was also used to get very sensitive measurements ofBMD. One vertebra and 1 femur were taken from a cohort of 4 wild typeand 8 homozygous mice. Measurements were taken of lumbar 5 vertebratrabecular bone volume, trabecular thickness, connectivity density andmidshaft femur total bone area and cortical thickness. The μCT40 scansprovided detailed information on bone mass and architecture. Multiplebones were placed into sample holders and scanned automatically.Instrument software was used to select regions of interest for analysis.Trabecular bone parameters were analyzed in the fifth lumbar vertebrae(LV5) at 16 micrometer resolution and cortical bone parameters wereanalyzed in the femur midshaft at a resolution of 20 micrometers.

Results:

DEXA: Both the male and female (−/−) mice exhibited decreased mean totaltissue mass and bone mineral content and density measurements whencompared with the historical means, the differences being more notablein the females. Female (−/−) mice showed decreased total body volumetricbone mineral density (vBMD), vertebrae bone mineral density (BMD), andtotal body bone mineral density (large difference >2SD). The (−/−) micealso showed decreased femur bone mineral density (BMD) and total bodybone mineral content (BMC).Micro CT: The male (−/−) mice exhibited decreased mean vertebraltrabecular bone volume, number, thickness, and connectivity density anddecreased mean femoral mid-shaft cortical thickness and cross-sectionalarea when compared with their gender-matched (+/+) littermates and thehistorical means.

The (−/−) mice analyzed by DEXA and bone micro CT analysis exhibiteddecreased bone measurements and decreased body mass measurements whencompared with their (+/+) littermates, suggestive of abnormal bonedisorders. The (−/−) mice exhibited a negative bone phenotype withabnormal decreased bone measurements reflective of bone metabolicdisorders. In addition, the decreased mean total tissue mass isindicative of a metabolic disorder related to tissue wasting disorders.The negative bone phenotype indicates that PRO290 polypeptides oragonists thereof would be useful for maintaining bone homeostasis. Inaddition, PRO290 polypeptides would be useful in bone healing or for thetreatment of arthritis or osteoporosis, whereas antagonists (orinhibitors) of PRO290 polypeptides or its encoding gene would lead toabnormal or pathological bone disorders including inflammatory diseasesassociated with abnormal bone metabolism including arthritis,osteoporosis and osteopenia.

(d) Phenotypic Analysis: Metabolism—Blood Chemistry

In the area of metabolism, targets may be identified for the treatmentof diabetes. Blood chemistry phenotypic analysis includes blood glucosemeasurements. The COBAS Integra 400 (mfr: Roche) was used for runningblood chemistry tests on the mice. In the area of metabolism, targetsmay be identified for the treatment of diabetes.

Results:

The female (−/−) mice also exhibited a notably decreased mean serumglucose level.

In these studies the mutant (−/−) mice showed a notably decreased serumglucose levels which could be due to an increased insulin sensitivity.Thus, antagonists (inhibitors) to PRO290 polypeptides or its encodinggene would be useful in the treatment of impaired glucose homeostasis.

(e) Phenotypic Analysis: Cardiology

In the area of cardiovascular biology, targets were identified hereinfor the treatment of hypertension, atherosclerosis, heart failure,stroke, various coronary artery diseases, dyslipidemias such as highcholesterol (hypercholesterolemia) and elevated serum triglycerides(hypertriglyceridemia), diabetes and/or obesity. The phenotypic testsincluded the measurement of serum cholesterol and triglycerides.

Blood Lipids

Procedure: A cohort of 4 wild type, 4 heterozygous and 8 homozygous micewere tested in this assay. High cholesterol levels and increasedtriglyceride blood levels are recognized risk factors in the developmentof cardiovascular disease and/or diabetes. Measuring blood lipidsfacilitates the finding of biological switches that regulate blood lipidlevels. Inhibition of factors which elevate blood lipid levels may beuseful for reducing the risk for cardiovascular disease. In these bloodchemistry tests, measurements were recorded using the COBAS Integra 400(mfr: Roche).

Results:

Blood Chemistry: The male (−/−) mice exhibited a decreased mean serumtriglyceride level when compared with that of their gender-matched (+/+)littermates and the historical mean.

In summary, these knockout mutant mice exhibited a positive phenotypewith regards to lipid metabolism. Thus, mutant mice deficient in thePRO290 gene can serve as a model for treatment of cardiovascular diseaseassociated with dyslipidemia, hypertension, atherosclerosis, heartfailure, stroke, or various coronary artery diseases.

48.5. Generation and Analysis of Mice Comprising DNA225543 (UNQ294) GeneDisruptions

In these knockout experiments, the gene encoding PRO36006 polypeptides(designated as DNA225543) (UNQ294) was disrupted. The gene specificinformation for these studies is as follows: the mutated mouse genecorresponds to nucleotide reference: M_(—)145581 ACCESSION: NM_(—)145581NID:21704167 Mus musculus sialic acid-binding lectin Siglec-F(LOC233186); protein reference: Q920G3 ACCESSION: Q920G3 NID: Musmusculus (Mouse). SIALIC ACID-BINDING LECTIN SIGLEC-F; the human genesequence reference: NM_(—)003830 ACCESSION: NM_(—)003830 NID:4502658Homo sapiens sialic acid binding Ig-like lectin 5 (SIGLEC5); the humanprotein sequence corresponds to reference: 015389 ACCESSION:015389 NID:Homo sapiens (Human). OB BINDING PROTEIN-2 (SIGLEC5).

The mouse gene of interest is Siglec5 (sialic acid binding Ig-likelectin 5), ortholog of human SIGLEC5. Aliases include mSiglec-F, sialicacid-binding lectin Siglec-F, OBBP2, CD33L2, OB-BP2, SIGLEC-5, CD33antigen-like 2, OB binding protein-2, and sialic acid-bindingimmunoglobulin-like lectin 5.

SIGLEC5 is a type I integral plasma membrane protein expressed primarilyon immature cells of the myelomonocytic lineage, particularly oneosinophils in blood and eosinophil precursors in bone marrow. Theprotein contains a signal peptide, four extracellularimmunoglobulin-like domains, a transmembrane segment, and one or twocytoplasmic immunoreceptor tyrosine-based inhibitory motifs (ITIMs).When tyrosine-phosphorylated, ITIMs can bind with SH2 domains of severalphosphatases. The extracellular domain of SIGLEC5 binds withalpha-2,3-linked sialic acid on lipopolysaccharides and likely functionsas a cell adhesion molecule or signal-transducing receptor (Cornish etal, Blood 92(6):2123-32 (1998); Patel et al, J Biol Chem274(32):22729-38 (1999); Angata et al, J Biol Chem 276(48):45128-36(2001); Zhang et al, Eur J Immunol 34(4):1175-84 (2004)). SIGLEC5 isexpressed on eosinophils, neutrophils, monocytes, lung, spleen, andplacenta (Zhang et al, Eur J Immunol 34(4):1175-84 (2004); Patel et al,J Biol Chem 274(32):22729-38 (1999); Erickson-Miller et al, Exp Hematol31(5):382-8 (2003)). Neutrophil SIGLEC5 expression is upregulated inresponse to treatment with fMLP and tumor necrosis factor-alpha, andSIGLEC5 is involved in augmenting neutrophil oxidative bursting inresponse to fMLP. These activities suggest that SIGLEC5 plays a role inimmune cell function by participating in cell-cell interactions orphagocytosis after exposure to microbes (Erickson-Miller et al, ExpHematol 31(5):382-8 (2003); Jones et al, Mol Microbiol 49(5):1213-25(2003)).

Targeted or gene trap mutations are generated in strain129SvEv^(Brd)-derived embryonic stem (ES) cells. The chimeric mice arebred to C57BL/6J albino mice to generate F1 heterozygous animals. Theseprogeny are intercrossed to generate F2 wild type, heterozygous, andhomozygous mutant progeny. On rare occasions, for example when very fewF1 mice are obtained from the chimera, F1 heterozygous mice are crossedto 129SvEv^(Brd)/C57 hybrid mice to yield additional heterozygousanimals for the intercross to generate the F2 mice. Level I phenotypicanalysis is performed on mice from this generation

wt het hom Total Observed 14 47 30 91 Expected 22.75 45.5 22.75 91Chi-Sq. = 1.61 Significance = 0.4470879 (hom/n) = 0.25 Avg. Litter Size= 9Mutation InformationMutation Type Homologous Recombination (standard)Description: Coding exons 1 through 4 and the preceding noncoding exonwere targeted (NCBI accession NM_(—)145581.1).1. Wild-type Expression Panel: Expression of the target gene wasdetected in brain, spinal cord, eye, thymus, spleen, lung, and kidneyamong the 13 adult tissue samples tested by RT-PCR.2. QC Expression: Disruption of the target gene was confirmed bySouthern hybridization analysis.

48.5.1. Phenotypic Analysis (for Disrupted Gene: DNA225543 (UNQ294)

(a) Overall Phenotypic Summary:

Mutation of the gene encoding the ortholog of human sialic acid bindingIg-like lectin 5 (SIGLEC5) resulted in an increased skin fibroblastproliferation rate in female (−/−) mice. In addition, the mutant (−/−)mice exhibited an increased percentage of B cells and B cell precursorsboth in the lymph nodes and in the peritoneal lavage. Gene disruptionwas confirmed by Southern blot.

(b) Immunology Phenotypic Analysis

Immune related and inflammatory diseases are the manifestation orconsequence of fairly complex, often multiple interconnected biologicalpathways which in normal physiology are critical to respond to insult orinjury, initiate repair from insult or injury, and mount innate andacquired defense against foreign organisms. Disease or pathology occurswhen these normal physiological pathways cause additional insult orinjury either as directly related to the intensity of the response, as aconsequence of abnormal regulation or excessive stimulation, as areaction to self, or as a combination of these.

Though the genesis of these diseases often involves multistep pathwaysand often multiple different biological systems/pathways, interventionat critical points in one or more of these pathways can have anameliorative or therapeutic effect. Therapeutic intervention can occurby either antagonism of a detrimental process/pathway or stimulation ofa beneficial process/pathway.

T lymphocytes (T cells) are an important component of a mammalian immuneresponse. T cells recognize antigens which are associated with aself-molecule encoded by genes within the major histocompatibilitycomplex (MHC). The antigen may be displayed together with MHC moleculeson the surface of antigen presenting cells, virus infected cells, cancercells, grafts, etc. The T cell system eliminates these altered cellswhich pose a health threat to the host mammal. T cells include helper Tcells and cytotoxic T cells. Helper T cells proliferate extensivelyfollowing recognition of an antigen-MHC complex on an antigen presentingcell. Helper T cells also secrete a variety of cytokines, i.e.,lymphokines, which play a central role in the activation of B cells,cytotoxic T cells and a variety of other cells which participate in theimmune response.

In many immune responses, inflammatory cells infiltrate the site ofinjury or infection. The migrating cells may be neutrophilic,eosinophilic, monocytic or lymphocytic as can be determined byhistological examination of the affected tissues. Current Protocols inImmunology, ed. John E. Coligan, 1994, John Wiley & Sons, Inc.

Many immune related diseases are known and have been extensivelystudied. Such diseases include immune-mediated inflammatory diseases(such as rheumatoid arthritis, immune mediated renal disease,hepatobiliary diseases, inflammatory bowel disease (IBD), psoriasis, andasthma), non-immune-mediated inflammatory diseases, infectious diseases,immunodeficiency diseases, neoplasia, and graft rejection, etc. In thearea of immunology, targets were identified for the treatment ofinflammation and inflammatory disorders.

In the area of immunology, targets have been identified herein for thetreatment of inflammation and inflammatory disorders. Immune relateddiseases, in one instance, could be treated by suppressing the immuneresponse. Using neutralizing antibodies that inhibit molecules havingimmune stimulatory activity would be beneficial in the treatment ofimmune-mediated and inflammatory diseases. Molecules which inhibit theimmune response can be utilized (proteins directly or via the use ofantibody agonists) to inhibit the immune response and thus ameliorateimmune related disease.

The following test was performed:

Fluorescence-Activated Cell-Sorting (FACS) Analysis

Procedure:

FACS analysis of immune cell composition from peripheral blood wasperformed including CD4, CD8 and T cell receptor to evaluate Tlymphocytes, CD19 for B lymphocytes, CD45 as a leukocyte marker and panNK for natural killer cells. The FACS analysis was carried out on 2 wildtype and 6 homozygous mice and included cells derived from thymus,spleen, bone marrow and lymph node.

In these studies, analyzed cells were isolated from thymus, peripheralblood, spleen, bone marrow and lymph nodes. Flow cytometry was designedto determine the relative proportions of CD4 and CD8 positive T cells, Bcells, NK cells and monocytes in the mononuclear cell population. ABecton-Dickinson FACS Calibur 3-laser FACS machine was used to assessimmune status. For Phenotypic Assays and Screening, this machine recordsCD4+/CD8−, CD8+/CD4−, NK, B cell and monocyte numbers in addition to theCD4+/CD8+ ratio.

The mononuclear cell profile was derived by staining a single sample oflysed peripheral blood from each mouse with a panel of sixlineage-specific antibodies: CD45 PerCP, anti-TCRb APC, CD4 PE, CD8FITC, pan-NK PE, and CD19 FITC. The two FITC and PE labeled antibodiesstain mutually exclusive cell types. The samples were analyzed using aBecton Dickinson FACSCalibur flow cytometer with CellQuest software.

Results:

Tissue Specific FACS-Project: The (−/−) mice exhibited an increasedpercentage of B cells in lymph node when compared with that of their(+/+) littermates. The (−/−) mice also exhibited a decreased percentageof B220− CD11b Hi cells and increased percentages of B220− CD11 Low andCD11b− cells in peritoneal lavage. Thus, it appears that UNQ294 is anegative regulator of B cell production and differentiation.

(c) Phenotypic Analysis: Metabolism—Blood Chemistry

In the area of metabolism, targets may be identified for the treatmentof diabetes. Blood chemistry phenotypic analysis includes blood glucosemeasurements. The COBAS Integra 400 (mfr: Roche) was used for runningblood chemistry tests on the mice. In the area of metabolism, targetsmay be identified for the treatment of diabetes.

Results:

Blood Chemistry: The female (−/−) mice exhibited an increased mean serumglucose level when compared with that of their gender-matched (+/+)littermates and the historical mean. However, all male (−/−) mice hadhigher than historical mean serum glucose levels.

Thus, the mutant (−/−) mice exhibited hyperglycemia which could beassociated with an altered glucose metabolism or diabetes.

(d) Adult Skin Cell Proliferation:

Procedure: Skin cells were isolated from 16 week old animals (2 wildtype and 4 homozygotes). These were developed into primary fibroblastcultures and the fibroblast proliferation rates were measured in astrictly controlled protocol. The ability of this assay to detecthyper-proliferative and hypo-proliferative phenotypes has beendemonstrated with p53 and Ku80. Proliferation was measured using Brduincorporation.

Specifically, in these studies the skin fibroblast proliferation assaywas used. An increase in the number of cells in a standardized culturewas used as a measure of relative proliferative capacity. Primaryfibroblasts were established from skin biopsies taken from wild type andmutant mice. Duplicate or triplicate cultures of 0.05 million cells wereplated and allowed to grow for six days. At the end of the cultureperiod, the number of cells present in the culture was determined usinga electronic particle counter.

Results:

Skin Proliferation: The female (−/−) mice exhibited an increased meanskin fibroblast proliferation rate when compared with that of theirgender-matched (+/+) littermates and the historical mean.

Thus, homozygous mutant mice demonstrated a hyper-proliferativephenotype. As suggested by these observations, PRO36006 polypeptides oragonists thereof could function as tumor suppressors and would be usefulin decreasing abnormal cell proliferation.

48.6. Generation and Analysis of Mice Comprising DNA45419-1252 (UNQ318)Gene Disruptions

In these knockout experiments, the gene encoding PRO363 polypeptides(designated as DNA45419-1252) (UNQ318) was disrupted. The gene specificinformation for these studies is as follows: the mutated mouse genecorresponds to nucleotide reference: NM_(—)133733 ACCESSION:NM_(—)133733 NID: gi 31542034 ref NM_(—)133733.2 Mus musculus RIKEN cDNA9030425E11 gene (9030425E11Rik); protein reference:Q8R373 ACCESSION:Q8R373NID: Mus musculus (Mouse). CAR-like membrane protein (Adipocyteadhesion molecule) (Mus musculus adult male cecum cDNA, RIKENfull-length enriched library, clone:9130232017product:ADIPOCYTE-SPECIFIC PROTEIN 5, full insert sequence); the humangene sequence reference: NM_(—)024769 Homo sapiens adipocyte-specificadhesion molecule (ASAM); the human protein sequence corresponds toreference: Q9H₆B4 ACCESSION: Q9H₆B4 NID: Homo sapiens (Human). cDNA:FLJ22415 FIS, CLONE HRC08561 (HYPOTHETICAL 41.3 KDA PROTEIN).

The mouse gene of interest is RIKEN cDNA 9030425E1 gene, ortholog ofhuman ASAM (adipocyte-specific adhesion molecule). Aliases include ASAM,CLMP, FLJ22415, asp5, IGSF11, CAR-like membrane protein, andadipocyte-specific protein 5.

ASAM is a type I plasma membrane protein that likely functions as a celladhesion molecule and tight junction component. The protein belongs tothe immunoglobulin superfamily, consisting of a signal peptide, animmunoglobulin-like domain, an immunoglobulin constant-2 type domain, atransmembrane segment, and a cytoplasmic tail. ASAM is expressedprimarily in epithelial cells from a wide variety of tissues and islikely to play a role cell-cell communication and tight junctionformation (Raschperger et al, J Biol Chem 279(1):796-804 (2004); Katohand Katoh, Int J Oncol 23(2):525-31 (2003)).

Targeted or gene trap mutations are generated in strain129SvEv^(Brd)-derived embryonic stem (ES) cells. The chimeric mice arebred to C57BL/6J albino mice to generate F1 heterozygous animals. Theseprogeny are intercrossed to generate F2 wild type, heterozygous, andhomozygous mutant progeny. On rare occasions, for example when very fewF1 mice are obtained from the chimera, F1 heterozygous mice are crossedto 129SvEv^(Brd)/C57 hybrid mice to yield additional heterozygousanimals for the intercross to generate the F2 mice. Level I phenotypicanalysis is performed on mice from this generation

wt het hom Total Observed 28 52 9 89 Expected 22.25 44.5 22.5 89 Chi-Sq.= 7.06 Significance = 0.029304916 (hom/n) = 0.17 Avg. Litter Size = 10Mutation InformationMutation Type Homologous Recombination (standard)1. Wild-type Expression Panel:2. QC Expression:

48.6.1. Phenotypic Analysis (for Disrupted Gene: DNA45419-1252 (UNQ318)

(a) Overall Phenotypic Summary:

Mutation of the gene encoding the ortholog of human adipocyte-specificadhesion molecule (ASAM) resulted in reduced viability of (−/−) mutants.Both CAT-Scan analysis and necropsy revealed bilateral hydronephrosisand inflammation in the surviving homozygous mutant mice, consistentwith the notably increased mean systolic blood pressure observedclinically as well as the increase in blood urea nitrogen. The mutant(−/−) mice showed “pear shaped abdomens”, as well as bilaterallyenlarged kidneys with chronic inflammation noted as polycystic kidneydisease. In addition, the mutants were smaller than their wild-typelittermates and exhibited numerous blood chemistry, immunological andneurological abnormalities. Further evidence of growth retardation isshown by mutant (−/−) mice also exhibiting decreased body fat, lean bodymass and total tissue mass with decreased bone mineral densitymeasurements. Disruption of the target gene was confirmed by Southernhybridization analysis.

(b) Pathology

Gross: Reduced viability of the (−/−) mice was observed.

Microscopic: At 12.5 days, there were 48 embryos observed: 11 (−/−)embryos, 18 (+/−) embryos, 10 (+/+) embryos, 5 resorption moles, and 4inconclusive.

All 3 (−/−) mice exhibited bilateral hydronephrosis. Suppurative andpyogranulomatous inflammation was also noted in 2/3 (−/−) mice,suggesting an increased susceptibility to bacterial infection in thesemutants (urine analysis showed a possible urinary tract infection).

(c) Immunology Phenotypic Analysis

Immune related and inflammatory diseases are the manifestation orconsequence of fairly complex, often multiple interconnected biologicalpathways which in normal physiology are critical to respond to insult orinjury, initiate repair from insult or injury, and mount innate andacquired defense against foreign organisms. Disease or pathology occurswhen these normal physiological pathways cause additional insult orinjury either as directly related to the intensity of the response, as aconsequence of abnormal regulation or excessive stimulation, as areaction to self, or as a combination of these.

Though the genesis of these diseases often involves multistep pathwaysand often multiple different biological systems/pathways, interventionat critical points in one or more of these pathways can have anameliorative or therapeutic effect. Therapeutic intervention can occurby either antagonism of a detrimental process/pathway or stimulation ofa beneficial process/pathway.

T lymphocytes (T cells) are an important component of a mammalian immuneresponse. T cells recognize antigens which are associated with aself-molecule encoded by genes within the major histocompatibilitycomplex (MHC). The antigen may be displayed together with MHC moleculeson the surface of antigen presenting cells, virus infected cells, cancercells, grafts, etc. The T cell system eliminates these altered cellswhich pose a health threat to the host mammal. T cells include helper Tcells and cytotoxic T cells. Helper T cells proliferate extensivelyfollowing recognition of an antigen-MHC complex on an antigen presentingcell. Helper T cells also secrete a variety of cytokines, i.e.,lymphokines, which play a central role in the activation of B cells,cytotoxic T cells and a variety of other cells which participate in theimmune response.

In many immune responses, inflammatory cells infiltrate the site ofinjury or infection. The migrating cells may be neutrophilic,eosinophilic, monocytic or lymphocytic as can be determined byhistological examination of the affected tissues. Current Protocols inImmunology, ed. John E. Coligan, 1994, John Wiley & Sons, Inc.

Many immune related diseases are known and have been extensivelystudied. Such diseases include immune-mediated inflammatory diseases(such as rheumatoid arthritis, immune mediated renal disease,hepatobiliary diseases, inflammatory bowel disease (IBD), psoriasis, andasthma), non-immune-mediated inflammatory diseases, infectious diseases,immunodeficiency diseases, neoplasia, and graft rejection, etc. In thearea of immunology, targets were identified for the treatment ofinflammation and inflammatory disorders.

In the area of immunology, targets have been identified herein for thetreatment of inflammation and inflammatory disorders. Immune relateddiseases, in one instance, could be treated by suppressing the immuneresponse. Using neutralizing antibodies that inhibit molecules havingimmune stimulatory activity would be beneficial in the treatment ofimmune-mediated and inflammatory diseases. Molecules which inhibit theimmune response can be utilized (proteins directly or via the use ofantibody agonists) to inhibit the immune response and thus ameliorateimmune related disease.

The following test was performed:

Hematology Analysis:

Test Description: Blood tests are carried out by Abbott's Cell-Dyn3500R, an automated hematology analyzer. Some of its features include afive-part WBC differential. ‘Patient’ reports can cover over 22parameters in all.

Results:

Hematology: The (−/−) mice exhibited an increased mean absoluteneutrophil count when compared with that of their (+/+) littermates andthe historical mean.

(d) Bone Metabolism & Body Diagnostics

(1) Tissue Mass & Lean Body Mass Measurements—Dexa

Dexa Analysis—Test Description:

Procedure: A cohort of wild type, heterozygous and homozygous mice weretested in this assay. Dual Energy X-ray Absorptiometry (DEXA) has beenused successfully to identify changes in total tissue mass (TTM).

The mouse was anesthetized by intraperitoneal injection of Avertin(1.25% 2,2,2,-tribromoethanol, 20 ml/kg body weight), body length andweight were measured, and then the mouse was placed in a prone positionon the platform of the PIXImus™ Densitometer (Lunar Inc.) for a DEXAscan. Using Lunar PIXImus software, the bone mineral density (BMD) andfat composition (% fat) and total tissue mass (TTM) were determined inthe regions of interest (ROI, i.e., whole body, vertebrae, and bothfemurs).

Body Measurements (Body Length & Weight):

Body Measurements: A measurement of body length and weight was performedat approximately 16 weeks of age.

Results:

Weight: The (−/−) mice exhibited decreased mean body weight whencompared with that of their gender-matched (+/+) littermates and thehistorical mean.

Length: The (−/−) mice exhibited decreased mean body length whencompared with that of their gender-matched (+/+) littermates and thehistorical mean.

In addition, 6 out of 8 (−/−) mice showed a “pear shaped” abdomen.

Fertility: The male (−/−) mouse available for analysis produced no pupsafter 40 days of breeding.

Basal Body Temperature: The male (−/−) mice exhibited a decreased medianbasal body temperature when compared with that of their gender-matched(+/+) littermates and the historical mean.

(2) Bone Metabolism: Radiology Phenotypic Analysis

In the area of bone metabolism, targets were identified herein for thetreatment of arthritis, osteoporosis, osteopenia and osteopetrosis aswell as identifying targets that promote bone healing. Tests included:

DEXA for measurement of bone mineral density on femur and vertebra

MicroCT for very high resolution and very high sensitivity measurementsof bone mineral density for both trabecular and cortical bone.

Dexa Analysis—Test Description:

Procedure: A cohort of wild type, heterozygous and homozygous weretested in this assay. Dual Energy X-ray Absorptiometry (DEXA) has beenused successfully to identify changes in bone. Anesthetized animals wereexamined and bone mineral content (BMC), BMC/LBM ratios, volumetric bonemineral density (vBMD), total body BMD, femur BMD and vertebra BMD weremeasured.

The mouse was anesthetized by intraperitoneal injection of Avertin(1.25% 2,2,2,-tribromoethanol, 20 ml/kg body weight), body length andweight were measured, and then the mouse was placed in a prone positionon the platform of the PIXImus™ Densitometer (Lunar Inc.) for a DEXAscan. Using Lunar PIXImus software, the bone mineral density (BMD) andfat composition (% fat) and total tissue mass (TTM) were determined inthe regions of interest (ROI) [i.e., whole body, vertebrae, and bothfemurs].

Bone MicroCT Analysis:

Procedure: MicroCT was also used to get very sensitive measurements ofBMD. One vertebra and 1 femur were taken from a cohort of wild type andhomozygous mice. Measurements were taken of lumbar 5 vertebra trabecularbone volume, trabecular thickness, connectivity density and midshaftfemur total bone area and cortical thickness. The μCT40 scans provideddetailed information on bone mass and architecture. Multiple bones wereplaced into sample holders and scanned automatically. Instrumentsoftware was used to select regions of interest for analysis. Trabecularbone parameters were analyzed in the fifth lumbar vertebrae (LV5) at 16micrometer resolution and cortical bone parameters were analyzed in thefemur midshaft at a resolution of 20 micrometers.

CATScan Analysis:

Test Description: Mouse was injected with a CT contrast agent, Omnipaque300 (Nycomed Amershan, 300 mg of iodine per ml, 0.25 ml per animal, or2.50-3.75 g iodine/kg of body weight) intraperitoneally. After restingin the cage for ˜10 minutes, the mouse was then sedated byintraperitoneal injection of Avertin (1.25% 2,2,2,-tribromoethanol, 20ml/kg body weight). A CAT-scan was performed using a MicroCAT scanner(ImTek, Inc.) with the anesthetized animal lying prone on the test bed.Three dimensional images were reconstructed by the Feldkamp algorithm ina cluster of workstations using an ImTek 3D RECON software.

Results:

DEXA: The male (−/−) mice exhibited notably decreased mean total tissuemass and lean body mass. Both the male and female (−/−) mice exhibitednotably decreased mean percent body fat, total fat mass, and all bonemineral density-related measurements when compared with those of theirgender-matched (+/+) littermates and the historical means.Micro CT: No notable difference. However, no (−/−) mice were availablefor analysis.CATScan: A113 (−/−) mice analyzed (M-154, F-81, and F-147) exhibitedbilaterally enlarged kidneys with marked inflammation, suggestingpolycystic kidney disease or severe hydronephrosis. These results areconsistent with the increased blood urea nitrogen levels and systemichypertension reported below. One (+/−) mice (F-76) also exhibitedmoderate hydronephrosis on the left side.

Mutant (−/−) mice deficient in the gene encoding PRO363 polypeptidesshow a phenotype consistent with growth retardation, marked by decreasedbody weight and length and tissue wasting diseases (decreased total bodyfat (%) and fat mass (g)). Thus, antagonists or inhibitors of PRO363polypeptides or its encoding gene would mimic these metabolic and growthrelated effects. On the other hand, PRO363 polypeptides or agoniststhereof would be useful in the prevention and/or treatment of suchmetabolic disorders as diabetes or other tissue wasting diseases.

In addition, the (−/−) mice analyzed by DEXA exhibited decreased bonemeasurements and decreased body mass measurements when compared withtheir (+/+) littermates, suggestive of abnormal bone disorders. The(−/−) mice exhibited a negative bone phenotype with abnormal decreasedbone measurements reflective of bone metabolic disorders. In addition,the decreased mean total tissue mass and lean body mass is indicative ofa metabolic disorder related to growth retardation and tissue wastingdisorders. The negative bone phenotype indicates that PRO363polypeptides or agonists thereof would be useful for maintaining bonehomeostasis in addition to normal growth development. In addition,PRO363 polypeptides would be useful in bone healing or for the treatmentof arthritis or osteoporosis, whereas antagonists (or inhibitors) ofPRO363 polypeptides or its encoding gene would lead to abnormal orpathological bone disorders including inflammatory diseases associatedwith abnormal bone metabolism including arthritis, osteoporosis andosteopenia.

(e) Phenotypic Analysis: CNS/Neurology

In the area of neurology, analysis focused herein on identifying in vivovalidated targets for the treatment of neurological and psychiatricdisorders including depression, generalized anxiety disorders, attentiondeficit hyperactivity disorder, obsessive compulsive disorder,schizophrenia, cognitive disorders, hyperalgesia and sensory disorders.Neurological disorders include the category defined as “anxietydisorders” which include but are not limited to: mild to moderateanxiety, anxiety disorder due to a general medical condition, anxietydisorder not otherwise specified, generalized anxiety disorder, panicattack, panic disorder with agoraphobia, panic disorder withoutagoraphobia, posttraumatic stress disorder, social phobia, specificphobia, substance-induced anxiety disorder, acute alcohol withdrawal,obsessive compulsive disorder, agoraphobia, bipolar disorder I or II,bipolar disorder not otherwise specified, cyclothymic disorder,depressive disorder, major depressive disorder, mood disorder,substance-induced mood disorder. In addition, anxiety disorders mayapply to personality disorders including but not limited to thefollowing types: paranoid, antisocial, avoidant behavior, borderlinepersonality disorders, dependent, histronic, narcissistic,obsessive-compulsive, schizoid, and schizotypal.

Procedure:

Behavioral screens were performed on a cohort of wild type, heterozygousand homozygous mutant mice. All behavioral tests were done between 12and 16 weeks of age unless reduced viability necessitates earliertesting. These tests included open field to measure anxiety, activitylevels and exploration.

Functional Observational Battery (FOB) Test—Tail Suspension Testing:

The FOB is a series of situations applied to the animal to determinegross sensory and motor deficits. A subset of tests from the Irwinneurological screen that evaluates gross neurological function is used.In general, short-duration, tactile, olfactory, and visual stimuli areapplied to the animal to determine their ability to detect and respondnormally. These simple tests take approximately 10 minutes and the mouseis returned to its home cage at the end of testing.

Tail Suspension Testing:

The tail suspension test is a procedure that has been developed as amodel for depressive-like behavior in rodents. In this particular setup,a mouse is suspended by its tail for 6 minutes, and in response themouse will struggle to escape from this position. After a certain periodof time the struggling of the mouse decreases and this is interpreted asa type of learned helplessness paradigm. Animals with invalid data (i.e.climbed their tail during the testing period) are excluded fromanalysis.

Results:

Tail Suspension2: The (−/−) mice exhibited increased median immobilitytime when compared with that of their (+/+) littermates and thehistorical mean, suggesting an increased depressive-like response in themutants.

Thus, knockout mice demonstrated a phenotype consistent with depression,generalized anxiety disorders, cognitive disorders, hyperalgesia andsensory disorders and/or bipolar disorders. Thus, PRO363 polypeptidesand agonists thereof would be useful for the treatment or ameliorationof the symptoms associated with depressive disorders.

(f) Phenotypic Analysis: Metabolism—Blood Chemistry

In the area of metabolism, targets may be identified for the treatmentof diabetes. Blood chemistry phenotypic analysis includes blood glucosemeasurements. The COBAS Integra 400 (mfr: Roche) was used for runningblood chemistry tests on the mice. In addition to measuring bloodglucose levels the following blood chemistry tests are also routinelyperformed: Alkaline Phosphatase; Alanine Amino-Transferase; Albumin;Bilirubin; Phosphorous; Creatinine; BUN=Blood Urea Nitrogen; Calcium;Uric Acid; Sodium; Potassium; and Chloride. In the area of metabolism,targets may be identified for the treatment of diabetes. Blood chemistryphenotypic analysis includes glucose tolerance tests to measure insulinsensitivity and changes in glucose metabolism. Abnormal glucosetolerance test results may indicate but may not be limited to thefollowing disorders or conditions: Diabetes Type 1 and Type 2, SyndromeX, various cardiovascular diseases and/or obesity.

Results:

Blood Chemistry: The male (−/−) mice exhibited an increased mean serumalkaline phosphatase level when compared with that of theirgender-matched (+/+) littermates and the historical mean. These resultsare consistent with the noted pathological findings of polycystic kidneydisease and increased blood urea nitrogen.

The (−/−) mice also exhibited a decreased mean serum glucose level whencompared with that of their gender-matched (+/+) littermates and thehistorical mean. In addition, both the male and female (−/−) miceexhibited an increased mean serum blood urea nitrogen level. Decreasedmean serum glucose levels is consistent with the observation of anenhanced glucose tolerance in these mutant (−/−) mice. Likewiseincreased blood urea nitrogen levels are consistent with kidneymalfunction and/or tissue wasting diseases.

(g) Phenotypic Analysis: Metabolism—Blood Chemistry/Glucose Tolerance

In the area of metabolism, targets may be identified for the treatmentof diabetes. Blood chemistry phenotypic analysis includes blood glucosemeasurements. The COBAS Integra 400 (mfr: Roche) was used for runningblood chemistry tests on the mice. In the area of metabolism, targetsmay be identified for the treatment of diabetes. Blood chemistryphenotypic analysis includes glucose tolerance tests to measure insulinsensitivity and changes in glucose metabolism. Abnormal glucosetolerance test results may indicate but may not be limited to thefollowing disorders or conditions: Diabetes Type 1 and Type 2, SyndromeX, various cardiovascular diseases and/or obesity.

Procedure: A cohort of wild type and homozygous mice were used in thisassay. The glucose tolerance test is the standard for defining impairedglucose homeostasis in mammals. Glucose tolerance tests were performedusing a Lifescan glucometer. Animals were injected IP at 2 g/kg withD-glucose delivered as a 20% solution and blood glucose levels weremeasured at 0, 30, 60 and 90 minutes after injection.

Results:

Oral Glucose Tolerance: The male (−/−) mouse available for analysisexhibited enhanced glucose tolerance when compared with that of itsgender-matched (+/+) littermates and the historical mean.

(h) Cardiology—Blood Pressure

Description:

Systolic blood pressure is measured via a noninvasive tail-cuff methodfor four days on the Visitech BP-2000 Blood Pressure Analysis System.The blood pressure is measured ten times each day for four days. Thefour days are then averaged to obtain a mouse's conscious systolic bloodpressure.

Results:

Blood Pressure: Both the male and female (−/−) mice exhibited notablyincreased mean systolic blood pressure when compared with that of theirgender-matched (+/+) littermates and the historical mean suggestingsystemic hypertension in the mutant (−/−) mice (consistent with thenoted kidney pathology).48.7. Generation and Analysis of Mice Comprising DNA46777-1253 (UNQ320)Gene Disruptions

In these knockout experiments, the gene encoding PRO365 polypeptides(designated as DNA46777-1253) (UNQ320) was disrupted. The gene specificinformation for these studies is as follows: the mutated mouse genecorresponds to nucleotide reference: NM_(—)020622 ACCESSION:NM_(—)020622 NID: gi 22296879 ref NM_(—)020622.1 Mus musculus RIKEN cDNA9030624C24 gene (9030624C24Rik); protein reference: Q9D309 ACCESSION:Q9D309 NID: Mus musculus (Mouse). Protein FAM3B precursor; the humangene sequence reference: NM_(—)058186 Homo sapiens family with sequencesimilarity 3, member B (FAM3B), transcript variant 1; the human proteinsequence corresponds to reference: P58499 ACCESSION:P58499 NID: Homosapiens (Human). Protein FAM3B precursor (Protein PRED44).

The mouse gene of interest is ORF9 (open reading frame 9), ortholog ofhuman FAM3B (family with sequence similarity 3, member B). Aliasesinclude 2-21, D16Jhu19e, 9030624C24Rik, PRED44, C21orf11, C21orf76,D21M16SJHU19e, cytokine-like protein 2-21, chromosome 21 open readingframe 11.

FAM3B is a secreted cytokine that likely functions as asignal-transducing ligand. The protein is expressed at high levels inalpha- and beta-cells of pancreatic islets and at lower levels in smallintestine, prostate, round spermatids within seminiferous tubules, nervecell bodies of numerous brain stem nuclei, and Purkinje cells of thecerebellum. FAM3B is capable of increasing basal levels of insulinsecretion from beta-cells and inducing apoptosis in islet cells by acaspase-3-mediated pathway (Zhu et al, Genomics 80(2): 144-50 (2002);Cao et al, Diabetes 52(9):2296-303 (2003)).

Targeted or gene trap mutations are generated in strain129SvEv^(Brd)-derived embryonic stem (ES) cells. The chimeric mice arebred to C57BL/6J albino mice to generate F1 heterozygous animals. Theseprogeny are intercrossed to generate F2 wild type, heterozygous, andhomozygous mutant progeny. On rare occasions, for example when very fewF1 mice are obtained from the chimera, F1 heterozygous mice are crossedto 129SvEv^(Brd)/C57 hybrid mice to yield additional heterozygousanimals for the intercross to generate the F2 mice. Level I phenotypicanalysis is performed on mice from this generation

wt het hom Total Observed 20 37 21 78 Expected 19.5 39 19.5 78 Chi-Sq. =0.91 Significance = 0.63444793 (hom/n) = 0.25 Avg. Litter Size = 8Mutation InformationMutation Type Homologous Recombination (standard)Description: Coding exons 1 and 2 were targeted (NCBI accessionNM_(—)020622.1).1. Wild-type Expression Panel: Expression of the target gene wasdetected in all 13 adult tissue samples tested by RT-PCR, except boneand adipose.2. QC Expression: Disruption of the target gene was confirmed bySouthern hybridization analysis.

48.7.1. Phenotypic Analysis (for Disrupted Gene: DNA46777-1253 (UNQ320)

(a) Overall Phenotypic Summary:

Mutation of the gene encoding the ortholog of human family with sequencesimilarity 3, member B (FAM3B) resulted in the (−/−) mice exhibiting anincrease percentage of CDb+CD11c− cells in the spleen (which containmonocytes/macrophages and neutrophils). The mutant (−/−) mice alsoexhibited a decreased mean heart rate. Gene disruption was confirmed bySouthern blot.

(b) Immunology Phenotypic Analysis

Immune related and inflammatory diseases are the manifestation orconsequence of fairly complex, often multiple interconnected biologicalpathways which in normal physiology are critical to respond to insult orinjury, initiate repair from insult or injury, and mount innate andacquired defense against foreign organisms. Disease or pathology occurswhen these normal physiological pathways cause additional insult orinjury either as directly related to the intensity of the response, as aconsequence of abnormal regulation or excessive stimulation, as areaction to self, or as a combination of these.

Though the genesis of these diseases often involves multistep pathwaysand often multiple different biological systems/pathways, interventionat critical points in one or more of these pathways can have anameliorative or therapeutic effect. Therapeutic intervention can occurby either antagonism of a detrimental process/pathway or stimulation ofa beneficial process/pathway.

T lymphocytes (T cells) are an important component of a mammalian immuneresponse. T cells recognize antigens which are associated with aself-molecule encoded by genes within the major histocompatibilitycomplex (MHC). The antigen may be displayed together with MHC moleculeson the surface of antigen presenting cells, virus infected cells, cancercells, grafts, etc. The T cell system eliminates these altered cellswhich pose a health threat to the host mammal. T cells include helper Tcells and cytotoxic T cells. Helper T cells proliferate extensivelyfollowing recognition of an antigen-MHC complex on an antigen presentingcell. Helper T cells also secrete a variety of cytokines, i.e.,lymphokines, which play a central role in the activation of B cells,cytotoxic T cells and a variety of other cells which participate in theimmune response.

In many immune responses, inflammatory cells infiltrate the site ofinjury or infection. The migrating cells may be neutrophilic,eosinophilic, monocytic or lymphocytic as can be determined byhistological examination of the affected tissues. Current Protocols inImmunology, ed. John E. Coligan, 1994, John Wiley & Sons, Inc.

Many immune related diseases are known and have been extensivelystudied. Such diseases include immune-mediated inflammatory diseases(such as rheumatoid arthritis, immune mediated renal disease,hepatobiliary diseases, inflammatory bowel disease (IBD), psoriasis, andasthma), non-immune-mediated inflammatory diseases, infectious diseases,immunodeficiency diseases, neoplasia, and graft rejection, etc. In thearea of immunology, targets were identified for the treatment ofinflammation and inflammatory disorders.

In the area of immunology, targets have been identified herein for thetreatment of inflammation and inflammatory disorders. Immune relateddiseases, in one instance, could be treated by suppressing the immuneresponse. Using neutralizing antibodies that inhibit molecules havingimmune stimulatory activity would be beneficial in the treatment ofimmune-mediated and inflammatory diseases. Molecules which inhibit theimmune response can be utilized (proteins directly or via the use ofantibody agonists) to inhibit the immune response and thus ameliorateimmune related disease.

The following test was performed:

Fluorescence-Activated Cell-Sorting (FACS) Analysis

Procedure:

FACS analysis of immune cell composition from peripheral blood wasperformed including CD4, CD8 and T cell receptor to evaluate Tlymphocytes, CD19 for B lymphocytes, CD45 as a leukocyte marker and panNK for natural killer cells. The FACS analysis was carried out on 2 wildtype and 6 homozygous mice and included cells derived from thymus,spleen, bone marrow and lymph node.

In these studies, analyzed cells were isolated from thymus, peripheralblood, spleen, bone marrow and lymph nodes. Flow cytometry was designedto determine the relative proportions of CD4 and CD8 positive T cells, Bcells, NK cells and monocytes in the mononuclear cell population. ABecton-Dickinson FACSCalibur 3-laser FACS machine was used to assessimmune status. For Phenotypic Assays and Screening, this machine recordsCD4+/CD8−, CD8+/CD4−, NK, B cell and monocyte numbers in addition to theCD4+/CD8+ ratio.

The mononuclear cell profile was derived by staining a single sample oflysed peripheral blood from each mouse with a panel of sixlineage-specific antibodies: CD45 PerCP, anti-TCRb APC, CD4 PE, CD8FITC, pan-NK PE, and CD19 FITC. The two FITC and PE labeled antibodiesstain mutually exclusive cell types. The samples were analyzed using aBecton Dickinson FACSCalibur flow cytometer with CellQuest software.

Results:

Tissue Specific FACS-Mouse: The (−/−) mice exhibited an increasedpercentage of CD11b+ CD11c− cells in spleen when compared with that oftheir (+/+) littermates. Thus, the mutant (−/−) mice exhibited elevatedlevels of monocytes/macrophages and neutrophils in the spleen.

(c) Cardiology—Heart Rate

Description:

Heart rate is measured via a noninvasive tail-cuff method for four dayson the Visitech BP-2000 Blood Pressure Analysis System. Heart rate ismeasured ten times each day for four days. The four days are thenaveraged to obtain a mouse's conscious heart rate.

Heart Rate: The (−/−) mice exhibited a decreased mean heart rate (1-2 SDbelow) when compared with that of their gender-matched (+/+) littermatesand the historical mean.

48.8 Generation and Analysis of Mice Comprising DNA45234-1277 (UNQ323)Gene Disruptions

In these knockout experiments, the gene encoding PRO382 polypeptides(designated as DNA45234-1277) (UNQ323) was disrupted. The gene specificinformation for these studies is as follows: the mutated mouse genecorresponds to nucleotide reference: NM_(—)080727 ACCESSION:NM_(—)080727 NID: 18141558 Mus musculus Mus musculus transmembraneprotease, serine 3 (Tmprss3); protein reference: Q8VDE0 ACCESSION:Q8VDE0 NID: Mus musculus (Mouse). TMPRSS3 PROTEIN; the human genesequence reference: NM_(—)024022 ACCESSION: NM_(—)024022 NID:13173470Homo sapiens transmembrane protease, serine 3 (TMPRSS3); the humanprotein sequence corresponds to reference: P57727 ACCESSION:P57727 NID:Homo sapiens (Human). TRANSMEMBRANE PROTEASE, SERINE 3 (EC 3.4.21.-)(SERINE PROTEASE TADG-12) (TUMOR ASSOCIATED DIFFERENTIALLY-EXPRESSEDGENE-12 PROTEIN).

The mouse gene of interest is Tmprss3 (transmembrane protease, serine3), ortholog of human TMPRS S3. Aliases include DFNB8, DFNB10, ECHOS1,TADG12, and serine protease TADG12.

TMPRSS3 is a type II integral membrane protein located primarily on theendoplasmic reticulum that likely functions as a channel-activatingserine protease. The protein contains a transmembrane segment, an LDLreceptor A domain, a scavenger receptor domain, a proteolytic activationsite, and a C-terminal serine protease domain (Wallrapp et al, CancerRes 60(10):2602-6 (2000); Guipponi et al, Hum Mol Genet 11(23):2829-36(2002)). Bioinformatic analyses (Clark et al, Genome Res 13(10):2265-70(2003)) suggest that TMPRSS3 may be an extracellular protein. TMPRSS3catalyzes the cleavage of epithelial amiloride-sensitive sodium channelENaC in vitro, activating the channel. TMPRSS3 is expressed in severaltissues that also express ENaC, such as the spiral ganglion, which arecells that support the organ of Corti and the stria vascularis in thecochlea. TMPRSS3 is also expressed inthymus, stomach, testis and E19mouse embryos. TMPRSS3 likely plays a role in hearing by regulating ENaCactivity, which maintains the low concentration of sodium in endolymphof the inner ear (Guipponi et al, Hum Mol Genet 11(23):2829-36 (2002)).TMPRSS3 is often over-expressed in certain types of cancer, where it mayplay a role in metastasis and tumor invasion (Wallrapp et al, Cancer Res60(10):2602-6 (2000); Underwood et al, Biochim Biophys Acta1502(3):337-50 (2000); Sawasaki et al, Tumour Biol 25(3):141-8 (2004)).Mutations in the TMPRSS3 gene can cause sensorineural deafness(Wattenhofer et al, J Mol Med 80(2): 124-31 (2002); Lee et al, J MedGenet 40(8):629-31 (2003); Ahmed et al, BMC Med Genet 5(1):24 (2004)).

Targeted or gene trap mutations are generated in strain129SvEv^(Brd)-derived embryonic stem (ES) cells. The chimeric mice arebred to C57BL/6J albino mice to generate F1 heterozygous animals. Theseprogeny are intercrossed to generate F2 wild type, heterozygous, andhomozygous mutant progeny. On rare occasions, for example when very fewF1 mice are obtained from the chimera, F1 heterozygous mice are crossedto 129SvEv^(Brd)/C57 hybrid mice to yield additional heterozygousanimals for the intercross to generate the F2 mice. Level I phenotypicanalysis is performed on mice from this generation

wt het hom Total Observed 16 48 18 82 Expected 20.5 41 20.5 82 Chi-Sq. =0.76 Significance = 0.68386143 (hom/n) = 0.24 Avg. Litter Size = 9Mutation InformationMutation Type: Homologous Recombination (standard)Description: Coding exon 1 was targeted (NCBI accession NM_(—)080727.1).1. Wild-type Expression Panel: Expression of the target gene wasdetected in brain, spinal cord, eye, thymus, spleen, and blood among the26 adult tissue samples tested by RT-PCR.2. QC Expression: Disruption of the target gene was confirmed bySouthern hybridization analysis.

48.8.1. Phenotypic Analysis (for Disrupted Gene: DNA45234-1277 (UNQ323)

(a) Overall Phenotypic Summary:

Mutation of the gene encoding the ortholog of human transmembraneprotease, serine 3 (TMPRSS3) resulted in no startle response in the(−/−) mice suggesting impaired hearing. Microscopic analysis revealeddegeneration of the Organ of Corti in the homozygous mutant mice,consistent with the hearing impairment noted during prepulse inhibitiontesting. Disruption of the target gene was confirmed by Southernhybridization analysis.

(b) Pathology

Microscopic: Of the 4 (−/−) mice available for analysis, 3 exhibiteddegeneration of the Organ of Corti; the organ was not in the level ofthe histological section in the remaining mutant. This is consistentwith the possible hearing impairment noted clinically.

(c) Phenotypic Analysis: CNS/Neurology

In the area of neurology, analysis focused herein on identifying in vivovalidated targets for the treatment of neurological and psychiatricdisorders including depression, generalized anxiety disorders, attentiondeficit hyperactivity disorder, obsessive compulsive disorder,schizophrenia, cognitive disorders, hyperalgesia and sensory disorders.Neurological disorders include the category defined as “anxietydisorders” which include but are not limited to: mild to moderateanxiety, anxiety disorder due to a general medical condition, anxietydisorder not otherwise specified, generalized anxiety disorder, panicattack, panic disorder with agoraphobia, panic disorder withoutagoraphobia, posttraumatic stress disorder, social phobia, specificphobia, substance-induced anxiety disorder, acute alcohol withdrawal,obsessive compulsive disorder, agoraphobia, bipolar disorder I or II,bipolar disorder not otherwise specified, cyclothymic disorder,depressive disorder, major depressive disorder, mood disorder,substance-induced mood disorder. In addition, anxiety disorders mayapply to personality disorders including but not limited to thefollowing types: paranoid, antisocial, avoidant behavior, borderlinepersonality disorders, dependent, histronic, narcissistic,obsessive-compulsive, schizoid, and schizotypal.

Procedure:

Behavioral screens were performed on a cohort of 4 wild type, 4heterozygous and 8 homozygous mutant mice. All behavioral tests weredone between 12 and 16 weeks of age unless reduced viabilitynecessitates earlier testing. These tests included open field to measureanxiety, activity levels and exploration.

Prepulse Inhibition of the Acoustic Startle Reflex

Prepulse inhibition of the acoustic startle reflex occurs when aloud 120decibel (dB) startle-inducing tone is preceded by a softer (prepulse)tone. The PPI paradigm consists of six different trial types (70 dBbackground noise, 120 dB alone, 74 dB+120 dB−pp4, 78 dB+120 dB−pp8, 82dB+120 dB−pp12, and 90 dB+120 dB−pp20) each repeated in pseudorandomorder six times for a total of 36 trials. The max response to thestimulus (V max) is averaged for each trial type. Animals with a 120 dBaverage value equal to or below 100 are excluded from analysis. Thepercent that the prepulse inhibits the animal's response to the startlestimulus is calculated and graphed.

Results:

PPI: All 8 (−/−) mice failed to exhibit a startle response, suggestingimpaired hearing in the mutants. Therefore, prepulse inhibition couldnot be assessed. Degeneration of the Organ of Corti is consistent withthese observations.

Circadian Test Description:

Female mice are individually housed at 4 pm on the first day of testingin 48.2 cm×26.5 cm home cages and administered food and water adlibitum. Animals are exposed to a 12-hour light/dark cycle with lightsturning on at 7 am and turning off at 7 pm. The system software recordsthe number of beam interruptions caused by the animal's movements, withbeam breaks automatically divided into ambulations. Activity is recordedin 60, one-hour intervals during the three-day test. Data generated aredisplayed by median activity levels recorded for each hour (circadianrhythm) and median total activity during each light/dark cycle(locomotor activity) over the three-day testing period.

Results:

Circadian: The female (−/−) mice exhibited a spike in activity in one ofthe light periods, but disruption of cycle was not consistent throughoutthe test period.

48.9. Generation and Analysis of Mice Comprising DNA26846-1397 (UNQ328)Gene Disruptions

In these knockout experiments, the gene encoding PRO444 polypeptides(designated as DNA26846-1397) (UNQ328) was disrupted. The gene specificinformation for these studies is as follows: the mutated mouse genecorresponds to nucleotide reference: NM_(—)026274 ACCESSION:NM_(—)026274 NID: gi 31541972 ref NM_(—)026274.2 Mus musculus RIKEN cDNA4930470D19 gene (4930470D19Rik); protein reference: Q8BVR6 ACCESSION:Q8BVR6NID: Mus musculus (Mouse). Mus musculus adult male testis cDNA,RIKEN full-length enriched library, clone:4930470D19product:hypothetical SPla and the RYanodine Receptor (SPRY)/SPRYdomain/RING finger containing protein, full insert sequence (RIKENcDNA4930470D19) (MKIAA1972 protein); the human gene sequence reference:NM_(—)133368 ACCESSION: NM_(—)133368 NID: gi 45387948 refNM_(—)133368.1Homo sapiens KIAA1972 protein (KIAA1972); the human protein sequencecorresponds to reference: Q96DX4 ACCESSION: Q96DX4 NID: Homo sapiens(Human). Hypothetical protein KIAA1972.

The mouse gene of interest is RIKEN cDNA 4930470D19 gene, ortholog ofhuman KIAA1972 protein.

KIAA1972 protein is a putative E3 ubiquitin ligase, containing a signalpeptide, a SPRY (splA and ryanodine receptor) domain (SMART accessionSM00449), and a RING (Ring finger) domain (SMART accession SM00184).RING domains often possess E3 ubiquitin ligase activity and can be foundin many E3 ubiquitin ligases (SMART accession SM00184). SPRY domains arelikely involved in protein-protein interactions (Wang et al, J Biol Chem280(16): 16393-401 (2005)). Bioinformatic analyses suggest that KIAA1972protein is extracellular (Clark et al, Genome Res 13(10):2265-70(2003)).

Targeted or gene trap mutations are generated in strain129SvEv^(Brd)-derived embryonic stem (ES) cells. The chimeric mice arebred to C57BL/6J albino mice to generate F1 heterozygous animals. Theseprogeny are intercrossed to generate F2 wild type, heterozygous, andhomozygous mutant progeny. On rare occasions, for example when very fewF1 mice are obtained from the chimera, F1 heterozygous mice are crossedto 129SvEv^(Brd)/C57 hybrid mice to yield additional heterozygousanimals for the intercross to generate the F2 mice. Level I phenotypicanalysis is performed on mice from this generation

wt het hom Total Observed 13 30 16 59 Expected 14.75 29.5 14.75 59Chi-Sq. = 0.83 Significance = 0.6603403 (hom/n) = 0.22 Avg. Litter Size= 8Mutation InformationMutation Type: Homologous Recombination (standard)Description: Coding exon 1 was targeted (NCBI accession NM_(—)026274.1).1. Wild-type Expression Panel: Expression of the target gene wasdetected in embryonic stem (ES) cells and in spinal cord; thymus;spleen; lung; liver; skeletal muscle; bone; stomach, small intestine,and colon; heart; adipose; asthmatic lung; and blood among the 26 adulttissue samples tested by RT-PCR.2. QC Expression: Disruption of the target gene was confirmed bySouthern hybridization analysis.

48.9.1. Phenotypic Analysis (for Disrupted Gene: DNA26846-1397 (UNQ328)

(a) Overall Phenotypic Summary:

Mutation of the gene encoding the ortholog of a putative human E3ubiquitin ligase (KIAA1972) resulted in small (−/−) mice that exhibitednumerous immunological abnormalities. The homozygous mutant mice weresmaller than their gender-matched wild-type littermates, exhibitingdecreased mean body weight and length, total tissue mass, and lean bodymass. Numerous immunological abnormalities were noted in the homozygousmutant mice, including a decreased percentage of natural killer cells inperipheral blood and an increased mean serum IL-6 and TNF alpha responseto LPS challenge when compared with that of their wild-type littermatesand the historical means. The immunological abnormalities observed inthe (−/−) mice could be due to mechanism of action mediated throughUNQ328's E3 ubiquitin ligase activity. In addition, the mutantsexhibited hypoactivity in open field testing or a depressive-likephenotype. The (−/−) mice showed systemic hypertension with an increaseddiastolic blood pressure and decreased heart rate. Blood chemistryresults showed increased alkaline phosphatase levels possibly related tohepatocellular dysfunction or biliary obstruction. The male (−/−) miceexhibited notably decreased micro CT vertebral bone densitymeasurements. Disruption of the target gene was confirmed by Southernhybridization analysis.

(b) Immunology Phenotypic Analysis

Immune related and inflammatory diseases are the manifestation orconsequence of fairly complex, often multiple interconnected biologicalpathways which in normal physiology are critical to respond to insult orinjury, initiate repair from insult or injury, and mount innate andacquired defense against foreign organisms. Disease or pathology occurswhen these normal physiological pathways cause additional insult orinjury either as directly related to the intensity of the response, as aconsequence of abnormal regulation or excessive stimulation, as areaction to self, or as a combination of these.

Though the genesis of these diseases often involves multistep pathwaysand often multiple different biological systems/pathways, interventionat critical points in one or more of these pathways can have anameliorative or therapeutic effect. Therapeutic intervention can occurby either antagonism of a detrimental process/pathway or stimulation ofa beneficial process/pathway.

T lymphocytes (T cells) are an important component of a mammalian immuneresponse. T cells recognize antigens which are associated with aself-molecule encoded by genes within the major histocompatibilitycomplex (MHC). The antigen may be displayed together with MHC moleculeson the surface of antigen presenting cells, virus infected cells, cancercells, grafts, etc. The T cell system eliminates these altered cellswhich pose a health threat to the host mammal. T cells include helper Tcells and cytotoxic T cells. Helper T cells proliferate extensivelyfollowing recognition of an antigen-MHC complex on an antigen presentingcell. Helper T cells also secrete a variety of cytokines, i.e.,lymphokines, which play a central role in the activation of B cells,cytotoxic T cells and a variety of other cells which participate in theimmune response.

In many immune responses, inflammatory cells infiltrate the site ofinjury or infection. The migrating cells may be neutrophilic,eosinophilic, monocytic or lymphocytic as can be determined byhistological examination of the affected tissues. Current Protocols inImmunology, ed. John E. Coligan, 1994, John Wiley & Sons, Inc.

Many immune related diseases are known and have been extensivelystudied. Such diseases include immune-mediated inflammatory diseases(such as rheumatoid arthritis, immune mediated renal disease,hepatobiliary diseases, inflammatory bowel disease (IBD), psoriasis, andasthma), non-immune-mediated inflammatory diseases, infectious diseases,immunodeficiency diseases, neoplasia, and graft rejection, etc. In thearea of immunology, targets were identified for the treatment ofinflammation and inflammatory disorders.

In the area of immunology, targets have been identified herein for thetreatment of inflammation and inflammatory disorders. Immune relateddiseases, in one instance, could be treated by suppressing the immuneresponse. Using neutralizing antibodies that inhibit molecules havingimmune stimulatory activity would be beneficial in the treatment ofimmune-mediated and inflammatory diseases. Molecules which inhibit theimmune response can be utilized (proteins directly or via the use ofantibody agonists) to inhibit the immune response and thus ameliorateimmune related disease.

The following tests were performed:

Acute Phase Response:

Test Description: Bacterial lipopolysaccharide (LPS) is an endotoxin,and as such is a potent inducer of an acute phase response and systemicinflammation. The Level I LPS mice were injected intraperitoneally(i.p.) with a sublethal dose of LPS in 200 μL sterile saline using a 26gauge needle. The doses were based on the average weight of the micetested at 1 μg/g body weight 3 hours after injection; a 100 ul bloodsample was then taken and analyzed for the presence of TNFa, MCP-1, andIL-6 on the FACSCalibur instrument.

Results:

Acute Phase Response: The (−/−) mice exhibited an increased mean serumIL-6 and TNFalpha response to LPS challenge when compared with that oftheir (+/+) littermates and the historical mean.

In summary, the LPS endotoxin challenge demonstrated that knockout micedeficient in the gene encoding PRO444 polypeptides exhibit immunologicalabnormalities when compared with their wild-type littermates. Inparticular, the mutant mice exhibited an increased ability to elicit animmunological response (IL-6 and TNFalpha production) when challengedwith the LPS endotoxin indicating a proinflammatory response. Il-6 andTNFalpha contribute to the later stages of B cell activation. Inaddition, IL-6 plays a critical role in inducing the acute phaseresponse and systemic inflammation. This suggests that inhibitors orantagonists to PRO444 polypeptides would stimulate the immune system andwould find utility in the cases wherein this effect would be beneficialto the individual such as in the case of leukemia, and other types ofcancer, and in immuno-compromised patients, such as AIDS sufferers.Accordingly, PRO444 polypeptides or agonists thereof would be useful ininhibiting the immune response and would be useful candidates forsuppressing harmful immune responses, e.g. in the case of graftrejection or graft-versus-host diseases.

Fluorescence-Activated Cell-Sorting (FACS) Analysis

Procedure:

FACS analysis of immune cell composition from peripheral blood wasperformed including CD4, CD8 and T cell receptor to evaluate Tlymphocytes, CD19 for B lymphocytes, CD45 as a leukocyte marker and panNK for natural killer cells. The FACS analysis was carried out on 2 wildtype and 6 homozygous mice and included cells derived from thymus,spleen, bone marrow and lymph node.

In these studies, analyzed cells were isolated from thymus, peripheralblood, spleen, bone marrow and lymph nodes. Flow cytometry was designedto determine the relative proportions of CD4 and CD8 positive T cells, Bcells, NK cells and monocytes in the mononuclear cell population. ABecton-Dickinson FACS Calibur 3-laser FACS machine was used to assessimmune status. For Phenotypic Assays and Screening, this machine recordsCD4+/CD8−, CD8+/CD4−, NK, B cell and monocyte numbers in addition to theCD4+/CD8+ ratio.

The mononuclear cell profile was derived by staining a single sample oflysed peripheral blood from each mouse with a panel of sixlineage-specific antibodies: CD45 PerCP, anti-TCRb APC, CD4 PE, CD8FITC, pan-NK PE, and CD19 FITC. The two FITC and PE labeled antibodiesstain mutually exclusive cell types. The samples were analyzed using aBecton Dickinson FACS Calibur flow cytometer with CellQuest software.

Results:

FACS3: The (−/−) mice exhibited an altered distribution of leukocytesubsets in the peripheral blood, characterized by a decreased meanpercentage of natural killer cells when compared with that of their(+/+) littermates and the historical mean.

These FACS results indicate that the homozygous mutant mice have adecreased mean percentage of natural killer cells. Natural killer cellsare the first line of defense to viral infection since these cells havebeen implicated in viral immunity and in defense against tumors. Naturalkiller cells or NK cells act as effectors in antibody-dependentcell-mediated cytotoxicity and have been identified by their ability tokill certain lymphoid tumor cell lines in vitro without the need forprior immunization or activation. Thus, PRO444 polypeptides or agoniststhereof, would be useful in stimulating or regulating this leukocyteproduction.

(c) Phenotypic Analysis: Metabolism—Blood Chemistry

In the area of metabolism, targets may be identified for the treatmentof diabetes. Blood chemistry phenotypic analysis includes blood glucosemeasurements. The COBAS Integra 400 (mfr: Roche) was used for runningblood chemistry tests on the mice. In addition to measuring bloodglucose levels the following blood chemistry tests are also routinelyperformed: Alkaline Phosphatase; Alanine Amino-Transferase; Albumin;Bilirubin; Phosphorous; Creatinine; BUN=Blood Urea Nitrogen; Calcium;Uric Acid; Sodium; Potassium; and Chloride. In the area of metabolism,targets may be identified for the treatment of diabetes. Blood chemistryphenotypic analysis includes glucose tolerance tests to measure insulinsensitivity and changes in glucose metabolism. Abnormal glucosetolerance test results may indicate but may not be limited to thefollowing disorders or conditions: Diabetes Type 1 and Type 2, SyndromeX, various cardiovascular diseases and/or obesity.

Results:

Blood Chemistry: Both the male and female (−/−) mice exhibited increasedmean serum alkaline phosphatase levels when compared with those of theirgender-matched (+/+) littermates and the historical means.

These results may be due to hepatocellular dysfunction or biliaryobstruction.

(d) Bone Metabolism & Body Diagnostics

(1) Tissue Mass & Lean Body Mass Measurements—Dexa

Dexa Analysis—Test Description:

Procedure: A cohort of 4 wild type, 4 heterozygous and 8 homozygous micewere tested in this assay. Dual Energy X-ray Absorptiometry (DEXA) hasbeen used successfully to identify changes in total tissue mass (TTM).

The mouse was anesthetized by intraperitoneal injection of Avertin(1.25% 2,2,2,-tribromoethanol, 20 ml/kg body weight), body length andweight were measured, and then the mouse was placed in a prone positionon the platform of the PIXImus™ Densitometer (Lunar Inc.) for a DEXAscan. Using Lunar PIXImus software, the bone mineral density (BMD) andfat composition (% fat) and total tissue mass (TTM) were determined inthe regions of interest (ROI, i.e., whole body, vertebrae, and bothfemurs).

Body Measurements (Body Length & Weight):

Body Measurements: A measurement of body length and weight was performedat approximately 16 weeks of age.

Results:

Weight: The (−/−) mice exhibited decreased mean body weight whencompared with that of their gender-matched (+/+) littermates and thehistorical means.

Length: The (−/−) mice exhibited decreased mean body length whencompared with that of their gender-matched (+/+) littermates and thehistorical means.

(2) Bone Metabolism: Radiology Phenotypic Analysis

In the area of bone metabolism, targets were identified herein for thetreatment of arthritis, osteoporosis, osteopenia and osteopetrosis aswell as identifying targets that promote bone healing. Tests included:

DEXA for measurement of bone mineral density on femur and vertebra

MicroCT for very high resolution and very high sensitivity measurementsof bone mineral density for both trabecular and cortical bone.

Dexa Analysis—Test Description:

Procedure: A cohort of 4 wild type, 4 heterozygous and 8 homozygous micewere tested in this assay. Dual Energy X-ray Absorptiometry (DEXA) hasbeen used successfully to identify changes in bone. Anesthetized animalswere examined and bone mineral content (BMC), BMC/LBM ratios, volumetricbone mineral density (vBMD), total body BMD, femur BMD and vertebra BMDwere measured.

The mouse was anesthetized by intraperitoneal injection of Avertin(1.25% 2,2,2,-tribromoethanol, 20 ml/kg body weight), body length andweight were measured, and then the mouse was placed in a prone positionon the platform of the PIXImus™ Densitometer (Lunar Inc.) for a DEXAscan. Using Lunar PIXImus software, the bone mineral density (BMD) andfat composition (% fat) and total tissue mass (TTM) were determined inthe regions of interest (ROI) [i.e., whole body, vertebrae, and bothfemurs].

Bone MicroCT Analysis:

Procedure: MicroCT was also used to get very sensitive measurements ofBMD. One vertebra and 1 femur were taken from a cohort of 4 wild typeand 8 homozygous mice. Measurements were taken of lumbar 5 vertebratrabecular bone volume, trabecular thickness, connectivity density andmidshaft femur total bone area and cortical thickness. The μCT40 scansprovided detailed information on bone mass and architecture. Multiplebones were placed into sample holders and scanned automatically.Instrument software was used to select regions of interest for analysis.Trabecular bone parameters were analyzed in the fifth lumbar vertebrae(LV5) at 16 micrometer resolution and cortical bone parameters wereanalyzed in the femur midshaft at a resolution of 20 micrometers.

Results:

DEXA: Both the male and female (−/−) mice exhibited decreased mean totaltissue mass, lean body mass, and bone mineral content and densitymeasurements when compared with those of their gender-matched (+/+)littermates and the historical means.

Micro CT: The male (−/−) mice exhibited notably decreased mean vertebraltrabecular bone volume, number, thickness, and connectivity density anddecreased mean femoral mid-shaft cortical thickness and cross-sectionalarea when compared with that of their gender-matched (+/+) littermatesand the historical means.

Mutant (−/−) mice deficient in the gene encoding PRO444 polypeptidesshow a phenotype consistent with growth retardation, marked by decreasedbody weight and length. Thus, antagonists or inhibitors of PRO444polypeptides or its encoding gene would mimic these metabolic and growthrelated effects. On the other hand, PRO444 polypeptides or agoniststhereof would be useful in the prevention and/or treatment of suchmetabolic disorders as diabetes or other tissue wasting diseases.

In addition, the (−/−) mice analyzed by DEXA and micro CT exhibiteddecreased bone measurements and decreased body mass measurements whencompared with their (+/+) littermates, suggestive of abnormal bonedisorders. The (−/−) mice exhibited a negative bone phenotype withabnormal decreased bone measurements reflective of bone metabolicdisorders. In addition, the decreased mean total tissue mass and leanbody mass is indicative of a metabolic disorder related to growthretardation and tissue wasting disorders. The negative bone phenotypeindicates that PRO444 polypeptides or agonists thereof would be usefulfor maintaining bone homeostasis in addition to normal growthdevelopment. In addition, PRO444 polypeptides would be useful in bonehealing or for the treatment of arthritis or osteoporosis, whereasantagonists (or inhibitors) of PRO444 polypeptides or its encoding genewould lead to abnormal or pathological bone disorders includinginflammatory diseases associated with abnormal bone metabolism includingarthritis, osteoporosis and osteopenia.

(e) Phenotypic Analysis: CNS/Neurology

In the area of neurology, analysis focused herein on identifying in vivovalidated targets for the treatment of neurological and psychiatricdisorders including depression, generalized anxiety disorders, attentiondeficit hyperactivity disorder, obsessive compulsive disorder,schizophrenia, cognitive disorders, hyperalgesia and sensory disorders.Neurological disorders include the category defined as “anxietydisorders” which include but are not limited to: mild to moderateanxiety, anxiety disorder due to a general medical condition, anxietydisorder not otherwise specified, generalized anxiety disorder, panicattack, panic disorder with agoraphobia, panic disorder withoutagoraphobia, posttraumatic stress disorder, social phobia, specificphobia, substance-induced anxiety disorder, acute alcohol withdrawal,obsessive compulsive disorder, agoraphobia, bipolar disorder I or II,bipolar disorder not otherwise specified, cyclothymic disorder,depressive disorder, major depressive disorder, mood disorder,substance-induced mood disorder. In addition, anxiety disorders mayapply to personality disorders including but not limited to thefollowing types: paranoid, antisocial, avoidant behavior, borderlinepersonality disorders, dependent, histronic, narcissistic,obsessive-compulsive, schizoid, and schizotypal.

Procedure:

Behavioral screens were performed on a cohort of 4 wild type, 4heterozygous and 8 homozygous mutant mice. All behavioral tests weredone between 12 and 16 weeks of age unless reduced viabilitynecessitates earlier testing. These tests included open field to measureanxiety, activity levels and exploration.

Open Field Test:

Several targets of known drugs have exhibited phenotypes in the openfield test. These include knockouts of the seratonin transporter, thedopamine transporter (Giros et al., Nature. 1996 Feb. 15;379(6566):606-12), and the GABA receptor (Homanics et al., Proc NatlAcad Sci USA. 1997 Apr. 15; 94(8):4143-8). An automated open-field assaywas customized to address changes related to affective state andexploratory patterns related to learning. First, the field (40×40 cm)was selected to be relatively large for a mouse, thus designed to pickup changes in locomotor activity associated with exploration. Inaddition, there were 4 holes in the floor to allow for nose-poking, anactivity specifically related to exploration. Several factors were alsodesigned to heighten the affective state associated with this test. Theopen-field test is the first experimental procedure in which the miceare tested, and the measurements that were taken were the subjects'first experience with the chamber. In addition, the open-field wasbrightly lit. All these factors will heighten the natural anxietyassociated with novel and open spaces. The pattern and extent ofexploratory activity, and especially the center-to-total distancetraveled ratio, may then be able to discern changes related tosusceptibility to anxiety or depression. A large arena (40 cm×40 cm,VersaMax animal activity monitoring system from AccuScan Instruments)with infrared beams at three different levels was used to recordrearing, hole poke, and locomotor activity. The animal was placed in thecenter and its activity was measured for 20 minutes. Data from this testwas analyzed in five, 4-minute intervals. The total distance traveled(cm), vertical movement number (rearing), number of hole pokes, and thecenter to total distance ratio were recorded.

The propensity for mice to exhibit normal habituation responses to anovel environment is assessed by determining the overall change in theirhorizontal locomotor activity across the 5 time intervals. Thiscalculated slope of the change in activity over time is determined usingnormalized, rather than absolute, total distance traveled. The slope isdetermined from the regression line through the normalized activity ateach of the 5 time intervals. Normal habituation is represented by anegative slope value.

Results:

The male (−/−) mice exhibited hypoactivity during open field testingwhen compared with their gender-matched (+/+) littermates and thehistorical mean, suggesting a decreased anxiety-like response in themutants.

A notable difference was observed during open field activity testing.The (−/−) mice exhibited an increased median sum time in the center(with hypoactivity) when compared with their gender-matched (+/+)littermates, which is indicative of a decreased anxiety-like response inthe mutants. Thus, knockout mice demonstrated a phenotype consistentwith depression, generalized anxiety disorders, cognitive disorders,hyperalgesia and sensory disorders and/or bipolar disorders. Thus,PRO444 polypeptides and agonists thereof would be useful for thetreatment or amelioration of the symptoms associated with depressivedisorders.

(f) Cardiology—Blood Pressure/Heart Rate

Description:

Systolic blood pressure is measured via a noninvasive tail-cuff methodfor four days on the Visitech BP-2000 Blood Pressure Analysis System.The blood pressure is measured ten times each day for four days. Thefour days are then averaged to obtain a mouse's conscious systolic bloodpressure.

Results:

Blood Pressure: The female (−/−) mice exhibited increased mean systolicblood pressure when compared with that of their gender-matched (+/+)littermates but within the historical mean.

Description:

Heart rate is measured via a noninvasive tail-cuff method for four dayson the Visitech BP-2000 Blood Pressure Analysis System. Heart rate ismeasured ten times each day for four days. The four days are thenaveraged to obtain a mouse's conscious heart rate.

Heart Rate: The female (−/−) mice exhibited a decreased mean heart ratewhen compared with that of their gender-matched (+/+) littermates andthe historical mean.

48.10. Generation and Analysis of Mice Comprising DNA50914-1289 (UNQ369)Gene Disruptions

In these knockout experiments, the gene encoding PRO705 polypeptides(designated as DNA50914-1289) (UNQ369) was disrupted. The gene specificinformation for these studies is as follows: the mutated mouse genecorresponds to nucleotide reference: NM_(—)011821 ACCESSION:NM_(—)011821 NID:7106324 Mus musculus Mus musculus glypican 6 (Gpc6);protein reference: Q9R087ACCESSION: Q9R087NID: Mus musculus (Mouse).GLYPICAN-6 PRECURSOR; the human gene sequence reference: NM_(—)005708ACCESSION: NM_(—)005708 NID:8051601 Homo sapiens glypican 6 (GPC6); thehuman protein sequence corresponds to reference: Q9Y625 ACCESSION:Q9Y625 NID: Homo sapiens (Human). GLYPICAN-6 PRECURSOR.

The mouse gene of interest is Gpc6 (glypican 6), ortholog of human GPC6.Aliases include MGC32221, 6720429C22Rik, bA62D23.1 (glypican 6),bA632L2.2 (glypican 6), and bA158B14.1 (glypican 6).

GPC6 is an extracellular glycosylphosphatidylinositol (GPI)-anchoredheparan sulfate proteoglycan that may function as a coreceptor orregulator of growth factor signaling. The protein is expressed in mosttissues, especially kidney and ovary. GPC6 likely plays a role inmorphogenesis during development (Veugelers et al, J Biol Chem274(38):26968-77 (1999); Paine-Saunders et al, Genomics 57(3):455-8(1999); De Cat and David, Semin Cell Dev Biol 12(2):117-25 (2001);Filmus, Glycobiology 11(3):19R-23R (2001)).

Targeted or gene trap mutations are generated in strain129SvEv^(Brd)-derived embryonic stem (ES) cells. The chimeric mice arebred to C57BL/6J albino mice to generate F1 heterozygous animals. Theseprogeny are intercrossed to generate F2 wild type, heterozygous, andhomozygous mutant progeny. On rare occasions, for example when very fewF1 mice are obtained from the chimera, F1 heterozygous mice are crossedto 129SvEv^(Brd)/C57 hybrid mice to yield additional heterozygousanimals for the intercross to generate the F2 mice. Level I phenotypicanalysis is performed on mice from this generation

wt het hom Total Observed 18 36 0 54 Expected 13.5 27 13.5 54 Chi-Sq. =12.36 Significance = 0.002070428 (hom/n) = 0.11 Avg. Litter Size = 8Mutation InformationMutation Type: Homologous Recombination (standard)Description: Coding exon 1 was targeted (NCBI accession NM_(—)011821.1).1. Wild-type Expression Panel: Expression of the target gene wasdetected in embryonic stem (ES) cells and in all 13 adult tissue samplestested by RT-PCR.2. QC Expression: Disruption of the target gene was confirmed bySouthern hybridization analysis.

48.10.1. Phenotypic Analysis (for Disrupted Gene: DNA50914-1289 (UNQ369)

(a) Overall Phenotypic Summary:

Mutation of the gene encoding the ortholog of human glypican 6 (GPC6)resulted in lethality of (−/−) mutants. However, even though all (−/−)mice showed embryonic lethality, the lethality was variable: some lethalbefore 12.5 days, some normal at 12.5 days. This observed lethality inthe homozygous mice could possibly be due to defective or lack of growthfactor signaling. Heterozygous (+/−) mice exhibited increased mean serumglucose levels as well as an increased platelet count. Gene disruptionwas confirmed by Southern blot.

(b) Pathology

Microscopic: At 12.5 days, 40 embryos were observed: 6 (−/−) embryos, 18(+/−) embryos, 11 (+/+) embryos, 4 resorption moles, and 1 inconclusive.However, no structural developmental abnormalities were detected in the(−/−) embryos.

Gene Expression: LacZ activity was not detected in the panel of tissuesby immunohistochemical analysis.

Discussion Related to Embryonic Developmental Abnormality of Lethality:

Embryonic lethality in knockout mice usually results from variousserious developmental problems including but not limited toneuro-degenerative diseases, angiogenic disorders, inflammatorydiseases, or where the gene/protein has an important role in basic cellsignaling processes in many cell types. In addition, embryonic lethalsare useful as potential cancer models. Likewise, the correspondingheterozygous (+/−) mutant animals are particularly useful when theyexhibit a phenotype and/or a pathology report which reveals highlyinformative clues as to the function of the knocked-out gene. Forinstance, EPO knockout animals were embryonic lethals, but the pathologyreports on the embryos showed a profound lack of RBCs.

(c) Immunology Phenotypic Analysis

Immune related and inflammatory diseases are the manifestation orconsequence of fairly complex, often multiple interconnected biologicalpathways which in normal physiology are critical to respond to insult orinjury, initiate repair from insult or injury, and mount innate andacquired defense against foreign organisms. Disease or pathology occurswhen these normal physiological pathways cause additional insult orinjury either as directly related to the intensity of the response, as aconsequence of abnormal regulation or excessive stimulation, as areaction to self, or as a combination of these.

Though the genesis of these diseases often involves multistep pathwaysand often multiple different biological systems/pathways, interventionat critical points in one or more of these pathways can have anameliorative or therapeutic effect. Therapeutic intervention can occurby either antagonism of a detrimental process/pathway or stimulation ofa beneficial process/pathway.

T lymphocytes (T cells) are an important component of a mammalian immuneresponse. T cells recognize antigens which are associated with aself-molecule encoded by genes within the major histocompatibilitycomplex (MHC). The antigen may be displayed together with MHC moleculeson the surface of antigen presenting cells, virus infected cells, cancercells, grafts, etc. The T cell system eliminates these altered cellswhich pose a health threat to the host mammal. T cells include helper Tcells and cytotoxic T cells. Helper T cells proliferate extensivelyfollowing recognition of an antigen-MHC complex on an antigen presentingcell. Helper T cells also secrete a variety of cytokines, i.e.,lymphokines, which play a central role in the activation of B cells,cytotoxic T cells and a variety of other cells which participate in theimmune response.

In many immune responses, inflammatory cells infiltrate the site ofinjury or infection. The migrating cells may be neutrophilic,eosinophilic, monocytic or lymphocytic as can be determined byhistological examination of the affected tissues. Current Protocols inImmunology, ed. John E. Coligan, 1994, John Wiley & Sons, Inc.

Many immune related diseases are known and have been extensivelystudied. Such diseases include immune-mediated inflammatory diseases(such as rheumatoid arthritis, immune mediated renal disease,hepatobiliary diseases, inflammatory bowel disease (IBD), psoriasis, andasthma), non-immune-mediated inflammatory diseases, infectious diseases,immunodeficiency diseases, neoplasia, and graft rejection, etc. In thearea of immunology, targets were identified for the treatment ofinflammation and inflammatory disorders.

In the area of immunology, targets have been identified herein for thetreatment of inflammation and inflammatory disorders. Immune relateddiseases, in one instance, could be treated by suppressing the immuneresponse. Using neutralizing antibodies that inhibit molecules havingimmune stimulatory activity would be beneficial in the treatment ofimmune-mediated and inflammatory diseases. Molecules which inhibit theimmune response can be utilized (proteins directly or via the use ofantibody agonists) to inhibit the immune response and thus ameliorateimmune related disease.

The following test was performed:

Hematology Analysis:

Test Description: Blood tests are carried out by Abbott's Cell-Dyn3500R, an automated hematology analyzer. Some of its features include afive-part WBC differential. ‘Patient’ reports can cover over 22parameters in all.

Results:

Hematology: The (+/−) mice exhibited an increased mean platelet countwhen compared with that of their (+/+) littermates and the historicalmean.

Thus, heterozygous mice resulted in a phenotype related to coagulationdisorders.

(d) Phenotypic Analysis: Metabolism—Blood Chemistry

In the area of metabolism, targets may be identified for the treatmentof diabetes. Blood chemistry phenotypic analysis includes blood glucosemeasurements. The COBAS Integra 400 (mfr: Roche) was used for runningblood chemistry tests on the mice. In the area of metabolism, targetsmay be identified for the treatment of diabetes.

Results:

Blood Chemistry: The female (+/−) mice exhibited an increased mean serumglucose level when compared with that of their gender-matched (+/+)littermates and the historical mean. Thus, the heterozygous (+/−) miceshowed a negative phenotype related to abnormal glucose metabolism.48.11. Generation and Analysis of Mice Comprising DNA58847-1383 (UNQ528)Gene Disruptions

In these knockout experiments, the gene encoding PRO1071 polypeptides(designated as DNA58847-1383) (UNQ528) was disrupted. The gene specificinformation for these studies is as follows: the mutated mouse genecorresponds to nucleotide reference: AK045085 Mus musculus 9.5 daysembryo parthenogenote cDNA, RIKEN full-length enriched library,clone:B130031C01 product:ADAM-TS RELATED PROTEIN 1 homolog [Homosapiens]; protein reference: Q8BLI0 ADAMTS-like protein 1 precursor(Punctin) gi|26337059|dbj|BAC32213.1| unnamed protein product [Musmusculus]; the human gene sequence reference: NM_(—)139238 Homo sapiensADAMTS-like 1 (ADAMTSL1), transcript variant 1; the human proteinsequence corresponds to reference: NP_(—)640329 ADAMTS-like 1 isoform 1[Homo sapiens].

The mouse gene of interest is Adamtsl1 (ADAMTS-like 1), ortholog ofhuman ADAMTSL1. Aliases include punctin-1, 6720426B09Rik, ADAMTSR1,MGC40193, punctin, ADAMTSL-1, ADAMTS-like, thrombospondin, and ADAM-TSrelated protein 1.

ADAMTSL1 is a secreted protein expressed primarily in skeletal musclethat likely functions as an extracellular matrix protein. ADAMTSL1 issimilar in structure to ADAMTS family of proteases. The protein containsa signal peptide, a thrombospondin type 1 repeat (PFAM accessionPF00090), an ADAM-TS spacer 1 region (PFAM accession PF05986), and twoto four more thrombospondin type I repeats but lacks metalloprotease anddisintegrin-like domains (Hirohata et al., J Biol Chem 277(14):12182-9(2002)).

Targeted or gene trap mutations are generated in strain129SvEv^(Brd)-derived embryonic stem (ES) cells. The chimeric mice arebred to C57BL/6J albino mice to generate F1 heterozygous animals. Theseprogeny are intercrossed to generate F2 wild type, heterozygous, andhomozygous mutant progeny. On rare occasions, for example when very fewF1 mice are obtained from the chimera, F1 heterozygous mice are crossedto 129SvEv^(Brd)/C57 hybrid mice to yield additional heterozygousanimals for the intercross to generate the F2 mice. Level I phenotypicanalysis is performed on mice from this generation

wt het hom Total Observed 23 35 17 75 Expected 18.75 37.5 18.75 75Chi-Sq. = 0.92 Significance = 0.63128364 (hom/n) = 0.24 Avg. Litter Size= 9Mutation InformationMutation Type: Homologous Recombination (standard)Description: Coding exon 1 was targeted (NCBI accession AK045085).1. Wild-type Expression Panel: Expression of the target gene wasdetected in all 26 adult tissue samples tested by RT-PCR, except bone,adipose, and blood.2. QC Expression: Disruption of the target gene was confirmed bySouthern hybridization analysis.

48.11.1. Phenotypic Analysis (for Disrupted Gene: DNA58847-1383 (UNQ528)

(a) Overall Phenotypic Summary:

Mutation of the gene encoding the ortholog of human ADAMTS-like 1(ADAMTSL1) resulted in (−/−) mice exhibiting a decreased mean skinfibroblast proliferation rate. In addition, the mutant (−/−) miceexhibited decreased total tissue mass, lean body mass and decreased bonemineral density measurements. The homozygous mice also showed elevatedmean serum glucose levels. Disruption of the target gene was confirmedby Southern hybridization analysis.

(b) Bone Metabolism & Body Diagnostics: Radiology Phenotypic Analysis

In the area of bone metabolism, targets were identified herein for thetreatment of arthritis, osteoporosis, osteopenia and osteopetrosis aswell as identifying targets that promote bone healing. Tests included:

DEXA for measurement of bone mineral density on femur and vertebra

MicroCT for very high resolution and very high sensitivity measurementsof bone mineral density for both trabecular and cortical bone.

Dexa Analysis—Test Description:

Procedure: A cohort of 4 wild type, 4 heterozygous and 8 homozygous micewere tested in this assay. Dual Energy X-ray Absorptiometry (DEXA) hasbeen used successfully to identify changes in bone. Anesthetized animalswere examined and bone mineral content (BMC), BMC/LBM ratios, volumetricbone mineral density (vBMD), total body BMD, femur BMD and vertebra BMDwere measured.

The mouse was anesthetized by intraperitoneal injection of Avertin(1.25% 2,2,2,-tribromoethanol, 20 ml/kg body weight), body length andweight were measured, and then the mouse was placed in a prone positionon the platform of the PIXImus™ Densitometer (Lunar Inc.) for a DEXAscan. Using Lunar PIXImus software, the bone mineral density (BMD) andfat composition (% fat) and total tissue mass (TTM) were determined inthe regions of interest (ROI) [i.e., whole body, vertebrae, and bothfemurs].

Bone MicroCT Analysis:

Procedure: MicroCT was also used to get very sensitive measurements ofBMD. One vertebra and 1 femur were taken from a cohort of 4 wild typeand 8 homozygous mice. Measurements were taken of lumbar 5 vertebratrabecular bone volume, trabecular thickness, connectivity density andmidshaft femur total bone area and cortical thickness. The μCT40 scansprovided detailed information on bone mass and architecture. Multiplebones were placed into sample holders and scanned automatically.Instrument software was used to select regions of interest for analysis.Trabecular bone parameters were analyzed in the fifth lumbar vertebrae(LV5) at 16 micrometer resolution and cortical bone parameters wereanalyzed in the femur midshaft at a resolution of 20 micrometers.

Results:

DEXA: The female (−/−) mice exhibited decreased mean total tissue mass,lean body mass, and bone mineral content and density measurements whencompared with those of their gender-matched (+/+) littermates and thehistorical means. However, the mean bone mineral content index (BMC/LBM)for the mutants was within the historical range.

Mutant (−/−) mice deficient in the gene encoding PRO1071 polypeptidesshow a phenotype consistent with tissue wasting diseases (decreasedtotal tissue mass and lean body mass). Thus, antagonists or inhibitorsof PRO1071 polypeptides or its encoding gene would mimic these metabolicrelated effects. On the other hand, PRO1071 polypeptides or agoniststhereof would be useful in the prevention and/or treatment of suchmetabolic disorders as cachexia or other tissue wasting diseases.

In addition, the (−/−) mice analyzed by DEXA exhibited decreased bonemeasurements and decreased body bone mineral content and densitymeasurements when compared with their (+/+) littermates, suggestive ofabnormal bone disorders. The (−/−) mice exhibited a negative bonephenotype with abnormal decreased bone measurements reflective of bonemetabolic disorders. The negative bone phenotype indicates that PRO1071polypeptides or agonists thereof would be useful for maintaining bonehomeostasis in addition to normal growth development. In addition,PRO1071 polypeptides would be useful in bone healing or for thetreatment of arthritis or osteoporosis, whereas antagonists (orinhibitors) of PRO1071 polypeptides or its encoding gene would lead toabnormal or pathological bone disorders including inflammatory diseasesassociated with abnormal bone metabolism including arthritis,osteoporosis and osteopenia.

(c) Phenotypic Analysis: Metabolism—Blood Chemistry

In the area of metabolism, targets may be identified for the treatmentof diabetes. Blood chemistry phenotypic analysis includes blood glucosemeasurements. The COBAS Integra 400 (mfr: Roche) was used for runningblood chemistry tests on the mice. In the area of metabolism, targetsmay be identified for the treatment of diabetes.

Results:

Blood Chemistry: The female (−/−) mice exhibited an increased mean serumglucose level when compared with that of their gender-matched (+/+)littermates and the historical mean.

Thus, the mutant (−/−) mice exhibited hyperglycemia which could beassociated with an altered glucose metabolism or diabetes. PRO1071polypeptides or agonists thereof would be useful in maintaining normalglucose levels/metabolism and possibly useful in the treatment ofdiabetes.

(d) Adult Skin Cell Proliferation:

Procedure: Skin cells were isolated from 16 week old animals (2 wildtype and 4 homozygotes). These were developed into primary fibroblastcultures and the fibroblast proliferation rates were measured in astrictly controlled protocol. The ability of this assay to detecthyper-proliferative and hypo-proliferative phenotypes has beendemonstrated with p53 and Ku80. Proliferation was measured using Brduincorporation.

Specifically, in these studies the skin fibroblast proliferation assaywas used. An increase in the number of cells in a standardized culturewas used as a measure of relative proliferative capacity. Primaryfibroblasts were established from skin biopsies taken from wild type andmutant mice. Duplicate or triplicate cultures of 0.05 million cells wereplated and allowed to grow for six days. At the end of the cultureperiod, the number of cells present in the culture was determined usinga electronic particle counter.

Results:

Skin Proliferation: The female (−/−) mice exhibited a decreased meanskin fibroblast proliferation rate when compared with that of theirgender-matched (+/+) littermates and the historical mean.

Thus, homozygous mutant mice demonstrated a hypo-proliferativephenotype. As suggested by these observations, antagonists or inhibitorsof PRO1071 polypeptides would mimic this hypo-proliferative phenotypeand could function as tumor suppressors and would be useful indecreasing abnormal cell proliferation.

48.12. Generation and Analysis of Mice Comprising DNA60619-1482 (UNQ563)Gene Disruptions

In these knockout experiments, the gene encoding PRO1125 polypeptides(designated as DNA60619-1482) (UNQ563) was disrupted. The gene specificinformation for these studies is as follows: the mutated mouse genecorresponds to nucleotide reference: NM_(—)013763 ACCESSION:NM_(—)013763 NID: gi 31543844 ref NM_(—)013763.2 Mus musculus transducin(beta)-like 2 (Tbl2); protein reference: Q8CFY0 ACCESSION: Q8CFY0 NID:Mus musculus (Mouse). Similar to transducin (Beta)-like 2; the humangene sequence reference: NM_(—)032988 ACCESSION: NM_(—)032988 NID: gi14670378 refNM_(—)032988.1 Homo sapiens transducin (beta)-like 2 (TBL2),transcript variant 2; the human protein sequence corresponds toreference: Q8N2L6 ACCESSION: Q8N2L6 NID: Homo sapiens (Human).Hypothetical protein FLJ90138.

The mouse gene of interest is Tbl2 (transducin [beta]-like 2), orthologof human TBL2. Aliases include WS-bTRP, WBSCR13, WS-betaTRP,DKFZP43N024, and Williams-Beuren syndrome chromosome region 13.

TBL2 is a putative secreted protein (Clark et al, Genome Res13(10):2265-70 (2003)), containing a signal peptide and five WD40repeats. Proteins with WD40 repeats generally function as scaffolds formulti-protein complex assembly. Examples of proteins with WD40 repeatsinclude G proteins, transcription factors, and E3 ubiquitin ligases(SMART accession SM00320). TBL2 is expressed primarily in testis,skeletal muscle, heart, and various endocrine tissues. TBL2 may beinvolved in Williams-Beuren syndrome (Meng et al, Hum Genet 103(5):590-9(1998); Perez-Jurado et al, Cytogenet Cell Genet 86(3-4):277-84 (1999)).

Targeted or gene trap mutations are generated in strain129SvEv^(Brd)-derived embryonic stem (ES) cells. The chimeric mice arebred to C57BL/6J albino mice to generate F1 heterozygous animals. Theseprogeny are intercrossed to generate F2 wild type, heterozygous, andhomozygous mutant progeny. On rare occasions, for example when very fewF1 mice are obtained from the chimera, F1 heterozygous mice are crossedto 129SvEv^(Brd)/C57 hybrid mice to yield additional heterozygousanimals for the intercross to generate the F2 mice. Level I phenotypicanalysis is performed on mice from this generation

wt het hom Total Observed 18 29 16 63 Expected 15.75 31.5 15.75 63Chi-Sq. = 3.19 Significance = 0.20290852 (hom/n) = 0.2 Avg. Litter Size= 8Mutation InformationMutation Type: Homologous Recombination (standard)Description: Coding exon 1 was targeted (NCBI accession NM_(—)013763.2).1. Wild-type Expression Panel: Expression of the target gene wasdetected in all 13 adult tissue samples tested by RT-PCR, exceptskeletal muscle; bone; stomach, small intestine, and colon; and adipose.2. QC Expression: Disruption of the target gene was confirmed bySouthern hybridization analysis.

48.12.1. Phenotypic Analysis (for Disrupted Gene: DNA60619-1482 (UNQ563)

(a) Overall Phenotypic Summary:

Mutation of the gene encoding the ortholog of human transducin(beta)-like 2 (TBL2) resulted in the mutant (−/−) mice exhibitingincreased microCT trabecular bone number and increased femoral mid-shaftcross-sectional area. The (−/−) mice also exhibited increased bodyweight and length compared with the (+/+) littermate controls. Male(−/−) mice showed fertility problems. Gene disruption was confirmed bySouthern blot.

(b) Bone Metabolism & Body Diagnostics

(1) Tissue Mass & Lean Body Mass Measurements—Dexa

Dexa Analysis—Test Description:

Procedure: A cohort of 4 wild type, 4 heterozygous and 8 homozygous micewere tested in this assay. Dual Energy X-ray Absorptiometry (DEXA) hasbeen used successfully to identify changes in total tissue mass (TTM).

The mouse was anesthetized by intraperitoneal injection of Avertin(1.25% 2,2,2,-tribromoethanol, 20 ml/kg body weight), body length andweight were measured, and then the mouse was placed in a prone positionon the platform of the PIXImus™ Densitometer (Lunar Inc.) for a DEXAscan. Using Lunar PIXImus software, the bone mineral density (BMD) andfat composition (% fat) and total tissue mass (TTM) were determined inthe regions of interest (ROI, i.e., whole body, vertebrae, and bothfemurs).

Body Measurements (Body Length & Weight):

Body Measurements: A measurement of body length and weight was performedat approximately 16 weeks of age.

Results:

Weight: The (−/−) mice exhibited increased mean body weight whencompared with that of their gender-matched (+/+) littermates and thehistorical mean, the difference being more notable in the males.

Length: The male (−/−) mice exhibited increased mean body length whencompared with that of their gender-matched (+/+) littermates and thehistorical mean.

Fertility:

Fertility: The male (−/−) mouse available for analysis produced no pupsafter 40 days of breeding with female (+/+) mice.

(2) Bone Metabolism: Radiology Phenotypic Analysis

In the area of bone metabolism, targets were identified herein for thetreatment of arthritis, osteoporosis, osteopenia and osteopetrosis aswell as identifying targets that promote bone healing. Tests included:

DEXA for measurement of bone mineral density on femur and vertebra

MicroCT for very high resolution and very high sensitivity measurementsof bone mineral density for both trabecular and cortical bone.

Dexa Analysis—Test Description:

Procedure: A cohort of 4 wild type, 4 heterozygous and 8 homozygous micewere tested in this assay. Dual Energy X-ray Absorptiometry (DEXA) hasbeen used successfully to identify changes in bone. Anesthetized animalswere examined and bone mineral content (BMC), BMC/LBM ratios, volumetricbone mineral density (vBMD), total body BMD, femur BMD and vertebra BMDwere measured.

The mouse was anesthetized by intraperitoneal injection of Avertin(1.25% 2,2,2,-tribromoethanol, 20 ml/kg body weight), body length andweight were measured, and then the mouse was placed in a prone positionon the platform of the PIXImus™ Densitometer (Lunar Inc.) for a DEXAscan. Using Lunar PIXImus software, the bone mineral density (BMD) andfat composition (% fat) and total tissue mass (TTM) were determined inthe regions of interest (ROI) [i.e., whole body, vertebrae, and bothfemurs].

Bone MicroCT Analysis:

Procedure: MicroCT was also used to get very sensitive measurements ofBMD. One vertebra and 1 femur were taken from a cohort of 4 wild typeand 8 homozygous mice. Measurements were taken of lumbar 5 vertebratrabecular bone volume, trabecular thickness, connectivity density andmidshaft femur total bone area and cortical thickness. The μCT40 scansprovided detailed information on bone mass and architecture. Multiplebones were placed into sample holders and scanned automatically.Instrument software was used to select regions of interest for analysis.Trabecular bone parameters were analyzed in the fifth lumbar vertebrae(LV5) at 16 micrometer resolution and cortical bone parameters wereanalyzed in the femur midshaft at a resolution of 20 micrometers.

Results:

Micro CT: The male (−/−) mice exhibited increased mean vertebraltrabecular bone number and increased mean femoral mid-shaftcross-sectional area when compared with that of their gender-matched(+/+) littermates and the historical means.

In summary, the (−/−) mice exhibited increased vertebral and femoralbone measurements when compared with their gender-matched (+/+)littermates. These results indicate that the knockout mutant phenotypemay be associated with such bone abnormalities as osteopetrosis.Osteopetrosis is a condition characterized by abnormal thickening andhardening of bone and abnormal fragility of the bones. As such, PRO1125polypeptides or agonists thereof would be beneficial for the treatmentof osteopetrosis or other osteo-related diseases. On the other hand,inhibitors or antagonists of PRO1125 polypeptides would be useful inbone healing.

In addition, the mutant (−/−) mice exhibited increased mean total tissuemass and lean body mass as well as increased body weight and length.These studies suggest that mutant (−/−) non-human transgenic animalsexhibit a negative phenotype that would be associated with obesity.Thus, PRO1125 polypeptides or agonists thereof are essential for normalgrowth and metabolic processes and especially would be important in theprevention and/or treatment of obesity.

48.13. Generation and Analysis of Mice Comprising DNA56865-1491 (UNQ572)Gene Disruptions

In these knockout experiments, the gene encoding PRO1134 polypeptides(designated as DNA56865-1491) (UNQ572) was disrupted. The gene specificinformation for these studies is as follows: the mutated mouse genecorresponds to nucleotide reference: NM_(—)029626 Mus musculus RIKENcDNA 2410004H05 gene (2410004H05Rik); protein reference: Q9CWT8 Q9CWT8Q9CWT8 2410004H05RIK PROTEIN; the human gene sequence reference:NM_(—)001010983 Homo sapiens glycosyltransferase 8 domain containing 1(GLT8D1), transcript variant 3; the human protein sequence correspondsto reference: Q9P0I5 Q9P0I5 Q9P0I5 AD-017 PROTEIN GLYCOSYLTRANSFERASE.

The mouse gene of interest is RIKEN cDNA 2410004H05 gene, ortholog ofhuman GLT8D1 (glycosyltransferase 8 domain containing 1). Aliasesinclude 5430414N14Rik, AD-017, MSTP139, FLJ14611, andglycosyltransferase AD-017.

GLT8D1 is a putative glycosyltransferase, consisting of a signal anchoror signal peptide and a glycosyltransferase family 8 domain. Enzymescontaining this domain typically catalyze the formation of O-glycosidicbonds between an acceptor molecule and an activated donor molecule. Anexample of an enzyme containing this domain is glycogenin, a proteintightly associated with glycogen synthase that catalyzes itsself-glucosylation using UDP-glucose as a cosubstrate (Pfam accessionPF01501). The cell location of GLT8D1 is ambiguous. Bioinformaticanalyses suggest that the protein may be extracellular or may be locatedon the Golgi apparatus (Coutinho et al., J Mol Biol 328(2):307-17(2003)).

Targeted or gene trap mutations are generated in strain129SvEv^(Brd)-derived embryonic stem (ES) cells. The chimeric mice arebred to C57BL/6J albino mice to generate F1 heterozygous animals. Theseprogeny are intercrossed to generate F2 wild type, heterozygous, andhomozygous mutant progeny. On rare occasions, for example when very fewF1 mice are obtained from the chimera, F1 heterozygous mice are crossedto 129SvEv^(Brd)/C57 hybrid mice to yield additional heterozygousanimals for the intercross to generate the F2 mice. Level I phenotypicanalysis is performed on mice from this generation

wt het hom Total Observed 23 41 19 83 Expected 20.75 41.5 20.75 83Chi-Sq. = 0.79 Significance = 0.67368 (hom/n) = 0.23 Avg. Litter Size =8Mutation InformationMutation Type Homologous Recombination (standard)Description: Coding exons 1 through 6 were targeted (NCBI accessionNM_(—)029626.1).1. Wild-type Expression Panel: Expression of the target gene wasdetected in embryonic stem (ES) cells and in all 26 adult tissue samplestested by RT-PCR.2. QC Expression: Disruption of the target gene was confirmed bySouthern hybridization analysis.

48.13.1. Phenotypic Analysis (for Disrupted Gene: DNA56865-1491 (UNQ572)

(a) Overall Phenotypic Summary:

Mutation of the gene encoding the ortholog of human glycosyltransferase8 domain containing 1 (GLT8D1) resulted in impaired sensorimotorgating/attention in male (−/−) mice. Gene disruption was confirmed bySouthern blot.

(b) Phenotypic Analysis: CNS/Neurology

In the area of neurology, analysis focused herein on identifying in vivovalidated targets for the treatment of neurological and psychiatricdisorders including depression, generalized anxiety disorders, attentiondeficit hyperactivity disorder, obsessive compulsive disorder,schizophrenia, cognitive disorders, hyperalgesia and sensory disorders.Neurological disorders include the category defined as “anxietydisorders” which include but are not limited to: mild to moderateanxiety, anxiety disorder due to a general medical condition, anxietydisorder not otherwise specified, generalized anxiety disorder, panicattack, panic disorder with agoraphobia, panic disorder withoutagoraphobia, posttraumatic stress disorder, social phobia, specificphobia, substance-induced anxiety disorder, acute alcohol withdrawal,obsessive compulsive disorder, agoraphobia, bipolar disorder I or II,bipolar disorder not otherwise specified, cyclothymic disorder,depressive disorder, major depressive disorder, mood disorder,substance-induced mood disorder. In addition, anxiety disorders mayapply to personality disorders including but not limited to thefollowing types: paranoid, antisocial, avoidant behavior, borderlinepersonality disorders, dependent, histronic, narcissistic,obsessive-compulsive, schizoid, and schizotypal.

Procedure:

Behavioral screens were performed on a cohort of 4 wild type, 4heterozygous and 8 homozygous mutant mice. All behavioral tests weredone between 12 and 16 weeks of age unless reduced viabilitynecessitates earlier testing. These tests included open field to measureanxiety, activity levels and exploration.

Prepulse Inhibition of the Acoustic Startle Reflex

Prepulse inhibition of the acoustic startle reflex occurs when a loud120 decibel (dB) startle-inducing tone is preceded by a softer(prepulse) tone. The PPI paradigm consists of six different trial types(70 dB background noise, 120 dB alone, 74 dB+120 dB−pp4, 78 dB+120dB−pp8, 82 dB+120 dB−pp12, and 90 dB+120 dB−pp20) each repeated inpseudo random order six times for a total of 36 trials. The max responseto the stimulus (V max) is averaged for each trial type. Animals with a120 dB average value equal to or below 100 are excluded from analysis.The percent that the prepulse inhibits the animal's response to thestartle stimulus is calculated and graphed.

Results:

PPI: The male (−/−) mice exhibited decreased inhibition during pp8,pp12, and pp20 when compared with that of their gender-matched (+/+)littermates and the historical means, suggesting impaired sensorimotorgating/attention in the mutants.

48.14. Generation and Analysis of Mice Comprising DNA59849-1504 (UNQ585)Gene Disruptions

In these knockout experiments, the gene encoding PRO1155 polypeptides(designated as DNA59849-1504) (UNQ585) was disrupted. The gene specificinformation for these studies is as follows: the mutated mouse genecorresponds to nucleotide reference: NM_(—)009312 ACCESSION:NM_(—)009312 NID:na Mus musculus tachykinin 2 (Tac2); protein reference:P55099 TKNK_MOUSE P55099 NEUROKININ B PRECURSOR NKB NEUROMEDI; the humangene sequence reference: NM_(—)013251 Homo sapiens tachykinin 3(neuromedin K, neurokinin beta) (TAC3); the human protein sequencecorresponds to reference: Q9UHF0 TKNK_HUMAN Q9UHF0 NEUROKININ BPRECURSOR NKB NEUROMEDI.

The mouse gene of interest is Tac2 (tachykinin 2), ortholog of humanTAC3 (tachykinin 3 [neuromedin K, neurokinin beta]). Aliases includesubstance K, neurokinin 2, neurokinin A, neuromedin L, neuropeptide K,neurokinin alpha, NKB, NKNB, PRO1155, ZNEUROK1, neuromedin K, neurokininbeta, gamma tachykinin 3, and neurokinin B-like protein.

TAC3 is a peptide neurotransmitter that functions as a ligand forreceptors TACR1, TACR2, or TACR3. TAC3 is released from peripheralneurons, interacting with its cognate receptors on a variety of tissues.TAC3 likely functions as paracrine hormone for regulation of vasculartone and blood pressure in the fetus and placenta. TAC3 dilates fetalvasculature and decreases fetal arterial blood pressure. TAC3 dilatesplacental vasculature by interacting with TACR1. Activation of TACR1 andconsequent decreases in blood pressure involve neither nitric oxidesynthesis nor prostacyclin synthesis (Brownbill et al, J Clin EndocrinolMetab 88(5):2164-70 (2003); Pinto et al, Eur J Pharmacol 494(2-3):233-9(2004)). TAC3 likely plays a role in maintenance of high placental bloodflow in normal pregnancy (Laliberte et al, Regul Pept 117(2):123-6(2004)), and TAC3 production may be involved in preeclampsia (Schlembachet al, Am J Obstet Gynecol 189(5):1418-22 (2003); Page et al, Nature405(6788):797-800 (2000)).

Targeted or gene trap mutations are generated in strain129SvEv^(Brd)-derived embryonic stem (ES) cells. The chimeric mice arebred to C57BL/6J albino mice to generate F1 heterozygous animals. Theseprogeny are intercrossed to generate F2 wild type, heterozygous, andhomozygous mutant progeny. On rare occasions, for example when very fewF1 mice are obtained from the chimera, F1 heterozygous mice are crossedto 129SvEv^(Brd)/C57 hybrid mice to yield additional heterozygousanimals for the intercross to generate the F2 mice. Level I phenotypicanalysis is performed on mice from this generation

wt het hom Total Observed 18 39 15 72 Expected 18 36 18 72 Chi-Sq. =1.15 Significance = 0.56270486 (hom/n) = 0.22 Avg. Litter Size = 9Mutation InformationMutation Type Homologous Recombination (standard)Description: The two noncoding exons preceding coding exon 1 and codingexon 1 were targeted (NCBI accession NM_(—)009312.1).1. Wild-type Expression Panel: Expression of the target gene wasdetected in brain, spinal cord, and eye among the 13 adult tissuesamples tested by RT-PCR.2. QC Expression: Disruption of the target gene was confirmed bySouthern hybridization analysis.

48.14.1. Phenotypic Analysis (for Disrupted Gene: DNA59849-1504 (UNQ585)

(a) Overall Phenotypic Summary:

Mutation of the gene encoding the ortholog of human tachykinin 3(neuromedin K, neurokinin beta) (TAC3) resulted in the (−/−) miceexhibiting decreased total body femur bone mineral density and bonemineral content and decreased connectivity density. Gene disruption wasconfirmed by Southern blot.

(b) Bone Metabolism: Radiology Phenotypic Analysis

In the area of bone metabolism, targets were identified herein for thetreatment of arthritis, osteoporosis, osteopenia and osteopetrosis aswell as identifying targets that promote bone healing. Tests included:

DEXA for measurement of bone mineral density on femur and vertebra

MicroCT for very high resolution and very high sensitivity measurementsof bone mineral density for both trabecular and cortical bone.

Dexa Analysis—Test Description:

Procedure: A cohort of 4 wild type, 4 heterozygous and 8 homozygous micewere tested in this assay. Dual Energy X-ray Absorptiometry (DEXA) hasbeen used successfully to identify changes in bone. Anesthetized animalswere examined and bone mineral content (BMC), BMC/LBM ratios, volumetricbone mineral density (vBMD), total body BMD, femur BMD and vertebra BMDwere measured.

The mouse was anesthetized by intraperitoneal injection of Avertin(1.25% 2,2,2,-tribromoethanol, 20 ml/kg body weight), body length andweight were measured, and then the mouse was placed in a prone positionon the platform of the PIXImus™ Densitometer (Lunar Inc.) for a DEXAscan. Using Lunar PIXImus software, the bone mineral density (BMD) andfat composition (% fat) and total tissue mass (TTM) were determined inthe regions of interest (ROI) [i.e., whole body, vertebrae, and bothfemurs].

Bone microCT Analysis:

Procedure: MicroCT was also used to get very sensitive measurements ofBMD. One vertebra and 1 femur were taken from a cohort of 4 wild typeand 8 homozygous mice. Measurements were taken of lumbar 5 vertebratrabecular bone volume, trabecular thickness, connectivity density andmidshaft femur total bone area and cortical thickness. The μCT40 scansprovided detailed information on bone mass and architecture. Multiplebones were placed into sample holders and scanned automatically.Instrument software was used to select regions of interest for analysis.Trabecular bone parameters were analyzed in the fifth lumbar vertebrae(LV5) at 16 micrometer resolution and cortical bone parameters wereanalyzed in the femur midshaft at a resolution of 20 micrometers.

Results:

DEXA: Female (−/−) mice exhibited decreased total body and femur bonemineral density as well as bone mineral content.

Micro CT: The (−/−) mice showed decreased connectivity density.

The (−/−) mice analyzed by DEXA and bone micro CT analysis exhibiteddecreased bone measurements when compared with their (+/+) littermates,suggestive of abnormal bone disorders. The (−/−) mice exhibited anegative bone phenotype with abnormal and decreased bone measurementsreflective of bone metabolic disorders. The negative bone phenotypeindicates that PRO1155 polypeptides or agonists thereof would be usefulfor maintaining bone homeostasis. In addition, PRO1155 polypeptideswould be useful in bone healing or for the treatment of arthritis orosteoporosis, whereas antagonists (or inhibitors) of PRO1155polypeptides or its encoding gene would lead to abnormal or pathologicalbone disorders including inflammatory diseases associated with abnormalbone metabolism including arthritis, osteoporosis and osteopenia.

48.15. Generation and Analysis of Mice Comprising DNA59820-1549 (UNQ651)Gene Disruptions

In these knockout experiments, the gene encoding PRO1281 polypeptides(designated as DNA59820-1549) (UNQ651) was disrupted. The gene specificinformation for these studies is as follows: the mutated mouse genecorresponds to nucleotide reference: NM_(—)001001566 Mus musculus DNAsegment, Chr 1, Brigham & Women's Genetics 1363 expressed (D1Bwg1363e);protein reference: Q6IQX7 ACCESSION: Q6IQX7 NID: Mus musculus (Mouse).Chondroitin polymerizing factor, isoform a; the human gene sequencerreference: NM_(—)024536 Homo sapiens chondroitin polymerizing factor(CHPF); the human protein sequence corresponds to reference: Q8IZ52ACCESSION: Q8IZ52 NID: Homo sapiens (Human). Chondroitin polymerizingfactor (Chondroitin sulfate synthase).

The mouse gene of interest is D1Bwg1363e (DNA segment, Chr 1, Brigham &Women's Genetics 1363 expressed), ortholog of human CHPF (chondroitinpolymerizing factor). Aliases include 1700028N03Rik, CSS2, FLJ22678, andchondroitin sulfate synthase 2.

CHPF is a putative type II membrane protein that functions as an enzymeor enzyme subunit involved in the biosynthesis of chondroitin sulfate, aproteoglycan found on cell surfaces and in extracellular matrix. Theprotein contains either a signal peptide or signal anchor and achondroitin N-acetylgalactosaminyltransferase domain (Pfam accessionPF05679). CHPF participates in catalyzing the polymerization ofalternating N-acetyl-D-galactosamine (GalNAc) and D-glucuronic acid(GlcUA) residues on chondroitin; however, it is not clear whether CHPFis capable of catalyzing this reaction alone or as a heterodimer withcarbohydrate (chondroitin) synthase 1. Like other chondroitin sulfatesynthesizing enzymes, CHPF is most likely located in the lumen of theGolgi apparatus (Kitagawa et al, J Biol Chem 278(26):23666-71 (2003);Yada et al, J Biol Chem 278(32):30235-47 (2003)). Bioinformaticanalyses, however, suggests that CHPF is an extracellular protein (Clarket al, Genome Res 13(10):2265-70 (2003)). CHPF plays a role inphysiological processes such as neural network formation, cellmigration, organogenesis, and cytokine signaling (Kitagawa et al, J BiolChem 278(26):23666-71 (2003); Yada et al, J Biol Chem 278(32):30235-47(2003); Izumikawa et al, J Biol Chem 279(51):53755-61 (2004)). Moreover,CHPF, like other chondroitin sulfate biosynthesizing enzymes, may be atarget for treatment of spinal cord injury (Kitagawa et al, J Biol Chem278(26):23666-71 (2003)).

Targeted or gene trap mutations are generated in strain129SvEv^(Brd)-derived embryonic stem (ES) cells. The chimeric mice arebred to C57BL/6J albino mice to generate F1 heterozygous animals. Theseprogeny are intercrossed to generate F2 wild type, heterozygous, andhomozygous mutant progeny. On rare occasions, for example when very fewF1 mice are obtained from the chimera, F1 heterozygous mice are crossedto 129SvEv^(Brd)/C57 hybrid mice to yield additional heterozygousanimals for the intercross to generate the F2 mice. Level I phenotypicanalysis is performed on mice from this generation

wt het hom Total Observed 15 30 18 63 Expected 15.75 31.5 15.75 63Chi-Sq. = 0.67 Significance = 0.71533805 (hom/n) = 0.27 Avg. Litter Size= 9Mutation InformationMutation Type: Homologous Recombination (standard)Description: Coding exons 1 through 3 as well as two noncoding exonspreceding coding exon 1 were targeted (NCBI accessionNM_(—)001001565.1).1. Wild-type Expression Panel: Expression of the target gene wasdetected in embryonic stem (ES) cells and in all 26 adult tissue samplestested by RT-PCR, except skeletal muscle and adipose.2. QC Expression: Disruption of the target gene was confirmed bySouthern hybridization analysis.

48.15.1. Phenotypic Analysis (for Disrupted Gene: DNA59820-1549 (UNQ651)

(a) Overall Phenotypic Summary:

Mutation of the gene encoding the ortholog of human chondroitinpolymerizing factor (CHPF) resulted in the (−/−) mice exhibitingincreased mean serum cholesterol and mean serum glucose levels. It isinteresting to note that UNQ651 is required for chondroitin synthetase(CHSY1)-mediated chondroitin polymerization and acts in chondroitinsulfate biosynthesis. CHSY1 appears to be develop spontaneous arthritis.Gene disruption was confirmed by Southern blot.

(b) Phenotypic Analysis: Cardiology

In the area of cardiovascular biology, targets were identified hereinfor the treatment of hypertension, atherosclerosis, heart failure,stroke, various coronary artery diseases, dyslipidemias such as highcholesterol (hypercholesterolemia) and elevated serum triglycerides(hypertriglyceridemia), diabetes and/or obesity. The phenotypic testsincluded the measurement of serum cholesterol and triglycerides.

Blood Lipids

Procedure: A cohort of 4 wild type, 4 heterozygous and 8 homozygous micewere tested in this assay. High cholesterol levels and increasedtriglyceride blood levels are recognized risk factors in the developmentof cardiovascular disease and/or diabetes. Measuring blood lipidsfacilitates the finding of biological switches that regulate blood lipidlevels. Inhibition of factors which elevate blood lipid levels may beuseful for reducing the risk for cardiovascular disease. In these bloodchemistry tests, measurements were recorded using the COBAS Integra 400(mfr: Roche).

Results:

Blood Chemistry: The male (−/−) mice exhibited an increased mean serumcholesterol level when compared with that of their gender-matched (+/+)littermates and the historical mean.

As summarized above, the (−/−) mice exhibited increased mean serumcholesterol levels when compared with their gender-matched (+/+)littermates and the historical means. Thus, mutant mice deficient in thePRO1281 gene may serve as a model for cardiovascular disease. PRO1281polypeptides or its encoding gene would be useful in regulating bloodlipids such as cholesterol. Thus, PRO1281 polypeptides or agoniststhereof would be useful in the treatment of such cardiovascular diseasesas hypertension, atherosclerosis, heart failure, stroke, variouscoronary diseases, hypercholesterolemia, diabetes and/or obesity.

(c) Phenotypic Analysis: Metabolism—Blood Chemistry

In the area of metabolism, targets may be identified for the treatmentof diabetes. Blood chemistry phenotypic analysis includes blood glucosemeasurements. The COBAS Integra 400 (mfr: Roche) was used for runningblood chemistry tests on the mice. In the area of metabolism, targetsmay be identified for the treatment of diabetes.

Results:

Both female heterozygous (+/−) and homozygous (−/−) mice exhibitedincreased mean serum glucose levels when compared with that of theirgender-matched (+/+) littermates and the historical mean.

Thus, the mutant (+/−) and (−/−) mice exhibited hyperglycemia whichcould be associated with an altered glucose metabolism or diabetes.PRO1281 polypeptides or agonists thereof would be useful in maintainingnormal glucose levels/metabolism and possibly useful in the treatment ofdiabetes.

48.16. Generation and Analysis of Mice Comprising DNA66675-1587 (UNQ698)Gene Disruptions

In these knockout experiments, the gene encoding PRO1343 polypeptides(designated as DNA66675-1587) (UNQ698) was disrupted. The gene specificinformation for these studies is as follows: the mutated mouse genecorresponds to nucleotide reference: NM_(—)172205 ACCESSION:NM_(—)172205 NID: gi 26251306 ref NM_(—)172205.1 Mus musculus suprabasin(Sbsn-pending); protein reference: 8CIT9 ACCESSION: Q8CIT9 NID: Musmusculus (Mouse). Suprabasal-specific protein suprabasin; the human genesequence reference: BC063640 Homo sapiens HLAR698, mRNA (cDNA cloneMGC:75533 IMAGE:4750640); the human protein sequence corresponds toreference: Q6UWP8 ACCESSION: Q6UWP8 NID: Homo sapiens (Human). HLAR698(Hypothetical protein).

The mouse gene of interest is Sbsn (suprabasin), ortholog of humanUNQ698 (HLAR698). Aliases include 1110005D19Rik and suprabasal-specificprotein.

Sbsn is a secreted protein expressed primarily in differentiatingsuprabasal keratinocytes that likely functions as a structural componentof skin. Sbsn contains a signal peptide and multiple regions of lowcomplexity, consisting of a large percentage of glycine, glutamine,histidine, and alanine residues. Sbsn is a substrate for tissuetransglutaminase 2 and epidermal transglutaminase 3, suggesting that theprotein can cross-link with extracellular and cellular components tobecome part of the epidermal cornified cell envelope. Sbsn is expressednot only in the suprabasal epithelium of epidermis but also in thesuprabasal epithelia of tongue and stomach. Sbsn likely plays a role indifferentiation of the epidermis and formation of the highly stratified,water impermeable barrier that is skin (Park et al, J Biol Chem277(47):45195-202 (2002); Moffatt et al, Gene 334:123-31 (2004)).

Targeted or gene trap mutations are generated in strain129SvEv^(Brd)-derived embryonic stem (ES) cells. The chimeric mice arebred to C57BL/6J albino mice to generate F1 heterozygous animals. Theseprogeny are intercrossed to generate F2 wild type, heterozygous, andhomozygous mutant progeny. On rare occasions, for example when very fewF1 mice are obtained from the chimera, F1 heterozygous mice are crossedto 129SvEv^(Brd)/C57 hybrid mice to yield additional heterozygousanimals for the intercross to generate the F2 mice. Level I phenotypicanalysis is performed on mice from this generation

wt het hom Total Observed 17 42 11 70 Expected 17.5 35 17.5 70 Chi-Sq. =0.38 Significance = 0.82695913 (hom/n) = 0.25 Avg. Litter Size = 8Mutation InformationMutation Type: Homologous Recombination (standard)Description: Coding exons 1 and 2 were targeted (NCBI accessionNM_(—)172205.1).1. Wild-type Expression Panel: Expression of the target gene wasdetected in embryonic stem (ES) cells and in all 13 adult tissue samplestested by RT-PCR, except liver, skeletal muscle, bone, and adipose.2. QC Expression: Disruption of the target gene was confirmed bySouthern hybridization analysis.

48.16.1. Phenotypic Analysis (for Disrupted Gene: DNA66675-1587 (UNQ698)

(a) Overall Phenotypic Summary:

Mutation of the gene encoding the ortholog of human UNQ698 (HLAR698)resulted in the (−/−) mice exhibiting decreased bone mineral densitymeasurements as well as elevated levels of mean serum cholesterol. Genedisruption was confirmed by Southern blot.

(b) Bone Metabolism & Body Diagnostics: Radiology Phenotypic Analysis

In the area of bone metabolism, targets were identified herein for thetreatment of arthritis, osteoporosis, osteopenia and osteopetrosis aswell as identifying targets that promote bone healing. Tests included:

DEXA for measurement of bone mineral density on femur and vertebra

MicroCT for very high resolution and very high sensitivity measurementsof bone mineral density for both trabecular and cortical bone.

Dexa Analysis—Test Description:

Procedure: A cohort of 4 wild type, 4 heterozygous and 8 homozygous micewere tested in this assay. Dual Energy X-ray Absorptiometry (DEXA) hasbeen used successfully to identify changes in bone. Anesthetized animalswere examined and bone mineral content (BMC), BMC/LBM ratios, volumetricbone mineral density (vBMD), total body BMD, femur BMD and vertebra BMDwere measured.

The mouse was anesthetized by intraperitoneal injection of Avertin(1.25% 2,2,2,-tribromoethanol, 20 ml/kg body weight), body length andweight were measured, and then the mouse was placed in a prone positionon the platform of the PIXImus™ Densitometer (Lunar Inc.) for a DEXAscan. Using Lunar PIXImus software, the bone mineral density (BMD) andfat composition (% fat) and total tissue mass (TTM) were determined inthe regions of interest (ROI) [i.e., whole body, vertebrae, and bothfemurs].

Results:

DEXA: The male (−/−) mice exhibited decreased total body bone mineraldensity and femur bone mineral density as well as total body volumetricbone mineral density measurements when compared with those of theirgender-matched (+/+) littermates and the historical means.

The (−/−) mice analyzed by DEXA exhibited decreased bone densitymeasurements when compared with their (+/+) littermates, suggestive ofabnormal bone disorders. The (−/−) mice exhibited a negative bonephenotype with abnormal decreased bone measurements reflective of bonemetabolic disorders. The negative bone phenotype indicates that PRO1343polypeptides or agonists thereof would be useful for maintaining bonehomeostasis in addition to normal growth development. In addition,PRO1343 polypeptides would be useful in bone healing or for thetreatment of arthritis or osteoporosis, whereas antagonists (orinhibitors) of PRO1343 polypeptides or its encoding gene would lead toabnormal or pathological bone disorders including inflammatory diseasesassociated with abnormal bone metabolism including arthritis,osteoporosis and osteopenia.

(c) Phenotypic Analysis: Cardiology

In the area of cardiovascular biology, targets were identified hereinfor the treatment of hypertension, atherosclerosis, heart failure,stroke, various coronary artery diseases, dyslipidemias such as highcholesterol (hypercholesterolemia) and elevated serum triglycerides(hypertriglyceridemia), diabetes and/or obesity. The phenotypic testsincluded the measurement of serum cholesterol and triglycerides.

Blood Lipids

Procedure: A cohort of 4 wild type, 4 heterozygous and 8 homozygous micewere tested in this assay. High cholesterol levels and increasedtriglyceride blood levels are recognized risk factors in the developmentof cardiovascular disease and/or diabetes. Measuring blood lipidsfacilitates the finding of biological switches that regulate blood lipidlevels. Inhibition of factors which elevate blood lipid levels may beuseful for reducing the risk for cardiovascular disease. In these bloodchemistry tests, measurements were recorded using the COBAS Integra 400(mfr: Roche).

Results:

Blood Chemistry: The female (−/−) mice exhibited increased mean serumcholesterol when compared with those of their gender-matched (+/+)littermates and the historical means.

As summarized above, the (−/−) mice exhibited increased mean serumcholesterol levels when compared with their gender-matched (+/+)littermates and the historical means. Thus, mutant mice deficient in thePRO1343 gene may serve as a model for cardiovascular disease. PRO1343polypeptides or its encoding gene would be useful in regulating bloodlipids such as cholesterol. Thus, PRO1343 polypeptides or agoniststhereof would be useful in the treatment of such cardiovascular diseasesas hypertension, atherosclerosis, heart failure, stroke, variouscoronary diseases, hypercholesterolemia, diabetes and/or obesity.

48.17. Generation and Analysis of Mice Comprising DNA59828-1608 (UNQ716)Gene Disruptions

In these knockout experiments, the gene encoding PRO1379 polypeptides(designated as DNA59828-1608) (UNQ716) was disrupted. The gene specificinformation for these studies is as follows: the mutated mouse genecorresponds to nucleotide reference: NM_(—)133779 ACCESSION:NM_(—)133779 NID: gi 19527005 ref NM_(—)133779.1 Mus musculus RIKEN cDNA4930534E15 gene (4930534E15Rik); protein reference: Q99JA3 ACCESSION:Q99JA3 NID: Mus musculus (Mouse). Neuronal development-associatedprotein (Neuronal development-associated protein 7) (RIKEN cDNA4930534E15 gene); the human gene sequence reference: NM_(—)015937ACCESSION: NM_(—)015937 NID: gi 23397652 refNM_(—)015937.2 Homo sapiensphosphatidyl inositol glycan class T (PIGT); the human protein sequencecorresponds to reference: Q969N2 ACCESSION: Q969N2 NID: Homo sapiens(Human). Phosphatidyl inositol glycan class T precursor (DJ453C12.7)(Hypothetical protein PLACE1010330).

The mouse gene of interest is Pigt (phosphatidylinositol glycan, classT), ortholog of human PIGT. Aliases include CGI-06, 4930534E15Rik,MGC8909, and GPI transamidase component PIG-T.

PIGT is a type I integral membrane protein located in the endoplasmicreticulum that likely functions as a noncatalytic subunit ofglycosylphosphatidylinositol (GPI) transamidase. GPI transamidaseconsists of five subunits and catalyzes the transfer of GPI to proteins.PIGT stabilizes the GPI transamidase complex and regulates access ofsubstrate proteins to the active sites (Ohishi et al, EMBO J.20(15):4088-98 (2001); Vainauskas et al, J Biol Chem 277(34):30535-42(2002); Ohishi et al, J Biol Chem 278(16):13959-67 (2003); Eisenhaber etal, Bioessays 25(4):367-85 (2003)).

Targeted or gene trap mutations are generated in strain129SvEv^(Brd)-derived embryonic stem (ES) cells. The chimeric mice arebred to C57BL/6J albino mice to generate F1 heterozygous animals. Theseprogeny are intercrossed to generate F2 wild type, heterozygous, andhomozygous mutant progeny. On rare occasions, for example when very fewF1 mice are obtained from the chimera, F1 heterozygous mice are crossedto 129SvEv^(Brd)/C57 hybrid mice to yield additional heterozygousanimals for the intercross to generate the F2 mice. Level I phenotypicanalysis is performed on mice from this generation

wt het hom Total Observed 15 30 0 45 Expected 11.25 22.5 11.25 45Chi-Sq. = 47.2 Significance = 5.6318367E−11 (hom/n) = 0.0 Avg. LitterSize = 7Mutation Type: Homologous Recombination (standard)Description: Coding exons 1 through 3 were targeted (NCBI accessionNM_(—)133779.1).1. Wild-type Expression Panel: Expression of the target gene wasdetected in embryonic stem (ES) cells and in all 26 adult tissue samplestested by RT-PCR.2. QC Expression: QC Images: Disruption of the target gene was confirmedby Southern hybridization analysis.

48.17.1. Phenotypic Analysis (for Disrupted Gene: DNA59828-1608 (UNQ716)

(a) Overall Phenotypic Summary:

UNQ716, DNA59828 Mutation of the gene encoding the ortholog of humanphosphatidylinositol glycan, class T (PIGT) resulted in lethality of(−/−) mice. Heterozygous (+/−) mice exhibited decreased bone mineralcontent and density measurements. Gene disruption was confirmed bySouthern blot.

(b) Pathology

Microscopic: Due to embryonic lethality, microscopic analysis was notperformed. At 12.5 days, there were 44 embryos observed: 27 (+/−)embryos, 5 (+/+) embryos, and 12 resorption moles.

Discussion Related to Embryonic Developmental Abnormality of Lethality:

Embryonic lethality in knockout mice usually results from variousserious developmental problems including but not limited toneurodegenerative diseases, angiogenic disorders, inflammatory diseases,or where the gene/protein has an important role in basic cell signalingprocesses in many cell types. In addition, embryonic lethals are usefulas potential cancer models. Likewise, the corresponding heterozygous(+/−) mutant animals are particularly useful when they exhibit aphenotype and/or a pathology report which reveals highly informativeclues as to the function of the knocked-out gene. For instance, EPOknockout animals were embryonic lethals, but the pathology reports onthe embryos showed a profound lack of RBCs.

(c) Bone Metabolism & Body Diagnostics: Radiology Phenotypic Analysis

In the area of bone metabolism, targets were identified herein for thetreatment of arthritis, osteoporosis, osteopenia and osteopetrosis aswell as identifying targets that promote bone healing. Tests included:

DEXA for measurement of bone mineral density on femur and vertebra

MicroCT for very high resolution and very high sensitivity measurementsof bone mineral density for both trabecular and cortical bone.

Dexa Analysis—Test Description:

Procedure: A cohort of 4 wild type, and 4 heterozygous mice were testedin this assay. Dual Energy X-ray Absorptiometry (DEXA) has been usedsuccessfully to identify changes in bone. Anesthetized animals wereexamined and bone mineral content (BMC), BMC/LBM ratios, volumetric bonemineral density (vBMD), total body BMD, femur BMD and vertebra BMD weremeasured.

The mouse was anesthetized by intraperitoneal injection of Avertin(1.25% 2,2,2,-tribromoethanol, 20 ml/kg body weight), body length andweight were measured, and then the mouse was placed in a prone positionon the platform of the PIXImus™ Densitometer (Lunar Inc.) for a DEXAscan. Using Lunar PIXImus software, the bone mineral density (BMD) andfat composition (% fat) and total tissue mass (TTM) were determined inthe regions of interest (ROI) [i.e., whole body, vertebrae, and bothfemurs].

Bone MicroCT Analysis:

Procedure: MicroCT was also used to get very sensitive measurements ofBMD. One vertebra and 1 femur were taken from a cohort of 4 wild typeand 8 heterozygous mice. Measurements were taken of lumbar 5 vertebratrabecular bone volume, trabecular thickness, connectivity density andmidshaft femur total bone area and cortical thickness. The μCT40 scansprovided detailed information on bone mass and architecture. Multiplebones were placed into sample holders and scanned automatically.Instrument software was used to select regions of interest for analysis.Trabecular bone parameters were analyzed in the fifth lumbar vertebrae(LV5) at 16 micrometer resolution and cortical bone parameters wereanalyzed in the femur midshaft at a resolution of 20 micrometers.

Results:

DEXA: The male (+/−) mice exhibited decreased mean bone mineral content(BMC), BMC/LBM, and total body bone mineral density (BMD) when comparedwith those of their gender-matched (+/+) littermates and the historicalmeans.

Micro CT: The male (+/−) mice exhibited decreased mean vertebraltrabecular bone volume, number, thickness, and connectivity density whencompared with those of their gender-matched (+/+) littermates and thehistorical means.

The (+/−) mice analyzed by DEXA and bone micro CT analysis exhibiteddecreased bone measurements when compared with their (+/+) littermates,suggestive of abnormal bone disorders. The (+/−) mice exhibited anegative bone phenotype with abnormal and decreased bone measurementsreflective of bone metabolic disorders. The negative bone phenotypeindicates that PRO1379 polypeptides or agonists thereof would be usefulfor maintaining bone homeostasis. In addition, PRO1379 polypeptideswould be useful in bone healing or for the treatment of arthritis orosteoporosis, whereas antagonists (or inhibitors) of PRO1379polypeptides or its encoding gene would lead to abnormal or pathologicalbone disorders including inflammatory diseases associated with abnormalbone metabolism including arthritis, osteoporosis and osteopenia.

48.18. Generation and Analysis of Mice Comprising DNA60740-1615 (UNQ717)Gene Disruptions

In these knockout experiments, the gene encoding PRO1380 polypeptides(designated as DNA60740-1615) (UNQ717) was disrupted. The gene specificinformation for these studies is as follows: the mutated mouse genecorresponds to nucleotide reference: NM_(—)023596 Mus musculus solutecarrier family 29 (nucleoside transporters), member 3 (Slc29a3); proteinreference: Q99P65 ACCESSION: Q99P65 NID: Mus musculus (Mouse).EQUILIBRATIVE NUCLEOSIDE TRANSPORTER 3; the human gene sequencereference: NM_(—)018344 Homo sapiens solute carrier family 29(nucleoside transporters), member 3 (SLC29A3); the human proteinsequence corresponds to reference: Q9BZD2 ACCESSION: Q9BZD2 NID: Homosapiens (Human). EQUILIBRATIVE NUCLEOSIDE TRANSPORTER 3.

The mouse gene of interest is Slc29a3 (solute carrier family 29[nucleoside transporters], member 3), ortholog of human SLC29A3. Aliasesinclude Ent3, 4933435C21Rik, FLJ1160, and equilibrative nucleosidetransporter 3.

SLC29A3 is a lysosomal integral membrane protein that functions as anequilibrative transporter, mediating the passive influx and efflux ofnucleosides (Baldwin et al, J Biol Chem 280(16): 15880-7 (2005)). Theprotein is expressed primarily in kidney and in Sertoli cells of thetestis (Lu et al, Drug Metab Dispos 32(12): 1455-61 (2004); Kato et al,J Pharmacol Exp Ther 312(2):601-8 (2005)). SLC29A3 may play a role inrelease of nucleosides produced by breakdown of nucleic acids inlysosomes. SLC29A3 may also play a role in the disposition of anticancerand antiviral nucleoside analogs (Lu et al, Drug Metab Dispos32(12):1455-61 (2004)) and in spermatogenesis (Kato et al, J PharmacolExp Ther 312(2):601-8 (2005)).

Targeted or gene trap mutations are generated in strain129SvEv^(Brd)-derived embryonic stem (ES) cells. The chimeric mice arebred to C57BL/6J albino mice to generate F1 heterozygous animals. Theseprogeny are intercrossed to generate F2 wild type, heterozygous, andhomozygous mutant progeny. On rare occasions, for example when very fewF1 mice are obtained from the chimera, F1 heterozygous mice are crossedto 129SvEv^(Brd)/C57 hybrid mice to yield additional heterozygousanimals for the intercross to generate the F2 mice. Level I phenotypicanalysis is performed on mice from this generation

wt het hom Total Observed 16 49 21 86 Expected 21.5 43 21.5 86 Chi-Sq. =5.5 Significance = 0.06392786 (hom/n) = 0.2 Avg. Litter Size = 10Mutation Type Homologous Recombination (standard)Description: Coding exons 1 and 2 were targeted (NCBI accessionNM_(—)023596.1).1. Wild-type Expression Panel: Expression of the target gene wasdetected in embryonic stem (ES) cells and in all 13 adult tissue samplestested by RT-PCR.2. QC Expression: QC Images: Disruption of the target gene was confirmedby Southern hybridization analysis.

48.18.1. Phenotypic Analysis (for Disrupted Gene: DNA60740-1615 (UNQ717)

(a) Overall Phenotypic Summary:

Mutation of the gene encoding the ortholog of human solute carrierfamily 29 (nucleoside transporters), member 3 (SLC29A3) resulted inhistiocytosis, lymphadenopathy, and hepatosplenomegaly in (−/−) micewith chronic inflammation. Necropsy revealed histiocytosis in the smallintestine, spleen, and lymph nodes of the homozygous mutant mice, alongwith lymphadenopathy and splenomegaly. UNQ717 gene is expressed onmonocytes and dendritic cells and is of special interest inimmunological disorders (with a possible role in autoimmunity). Thehomozygous mutant mice were also anemic and exhibited numerous otherimmunological and blood chemistry abnormalities when compared with thelevels for their wild-type littermates and the historical means. The(−/−) mice also exhibited a decreased stress induced hyperthermiaresponse compared with their littermate (+/+) controls. Disruption ofthe target gene was confirmed by Southern hybridization analysis.

(b) Pathology

Gross: The (−/−) mice exhibited splenomegaly and lymphadenopathy.

Microscopic: All 6 (−/−) mice analyzed exhibited varying degrees ofhistiocytosis in the small intestine, lymph nodes, and spleen. The (−/−)mice also exhibited lymphoid depletion (T cell) in the spleen, lymphnodes, and thymus. In the earliest stages of disease, there wasvacuolization of antigen presenting cells in the thymic cortex,associated with apoptosis of cortical thymocytes, and a minimalhistiocytic infiltrate in the jejunum. In more advanced cases, thespleen was mildly enlarged with multifocal replacement of T-cells(periarteriolar lymphoid sheaths) by histiocytic cells. The B cell areas(follicles) were relatively normal. There was increased erythropoiesisin the splenic red pulp in most mutants; however, scattered erythroidcells had dark shrunken nuclei surrounded by an expanded clearcytoplasm. Lymph nodes were enlarged and contained a diffuse histiocyticinfiltrate in the medullary cords and paracortical areas withconcomitant decreases in T cell lymphocytes. The submucosa and laminapropria of the shortened jejunal villi was expanded by a cell infiltrateconsisting primarily of histiocytes. The duodenum and ileum had similarbut milder infiltrates. The thymic cortex contained increased numbers ofbody macrophages with abundant highly vacuolated cytoplasm. In the 2most severe cases, there was marked enlargement of the spleen and alllymph nodes due to a massive infiltrate of histiocytic cells. The liversinusoids and small intestinal mucosa were also diffusely infiltrated byhistiocytes, and there was marked blunting and fusion of smallintestinal villi. Again, the most severe lesions were in the jejunum.Histiocyte infiltrates extended into the interstitium of the pancreas,resulting in atrophy and a loss of acinar glands. There was a markedgeneralized loss of T cells in all lymphoid tissues. The distribution ofthe histiocytic cells in tissues suggests that they may be of T celllineage.

(c) Immunology Phenotypic Analysis

Immune related and inflammatory diseases are the manifestation orconsequence of fairly complex, often multiple interconnected biologicalpathways which in normal physiology are critical to respond to insult orinjury, initiate repair from insult or injury, and mount innate andacquired defense against foreign organisms. Disease or pathology occurswhen these normal physiological pathways cause additional insult orinjury either as directly related to the intensity of the response, as aconsequence of abnormal regulation or excessive stimulation, as areaction to self, or as a combination of these.

Though the genesis of these diseases often involves multistep pathwaysand often multiple different biological systems/pathways, interventionat critical points in one or more of these pathways can have anameliorative or therapeutic effect. Therapeutic intervention can occurby either antagonism of a detrimental process/pathway or stimulation ofa beneficial process/pathway.

T lymphocytes (T cells) are an important component of a mammalian immuneresponse. T cells recognize antigens which are associated with aself-molecule encoded by genes within the major histocompatibilitycomplex (MHC). The antigen may be displayed together with MHC moleculeson the surface of antigen presenting cells, virus infected cells, cancercells, grafts, etc. The T cell system eliminates these altered cellswhich pose a health threat to the host mammal. T cells include helper Tcells and cytotoxic T cells. Helper T cells proliferate extensivelyfollowing recognition of an antigen-MHC complex on an antigen presentingcell. Helper T cells also secrete a variety of cytokines, i.e.,lymphokines, which play a central role in the activation of B cells,cytotoxic T cells and a variety of other cells which participate in theimmune response.

In many immune responses, inflammatory cells infiltrate the site ofinjury or infection. The migrating cells may be neutrophilic,eosinophilic, monocytic or lymphocytic as can be determined byhistological examination of the affected tissues. Current Protocols inImmunology, ed. John E. Coligan, 1994, John Wiley & Sons, Inc.

Many immune related diseases are known and have been extensivelystudied. Such diseases include immune-mediated inflammatory diseases(such as rheumatoid arthritis, immune mediated renal disease,hepatobiliary diseases, inflammatory bowel disease (IBD), psoriasis, andasthma), non-immune-mediated inflammatory diseases, infectious diseases,immunodeficiency diseases, neoplasia, and graft rejection, etc. In thearea of immunology, targets were identified for the treatment ofinflammation and inflammatory disorders.

In the area of immunology, targets have been identified herein for thetreatment of inflammation and inflammatory disorders. Immune relateddiseases, in one instance, could be treated by suppressing the immuneresponse. Using neutralizing antibodies that inhibit molecules havingimmune stimulatory activity would be beneficial in the treatment ofimmune-mediated and inflammatory diseases. Molecules which inhibit theimmune response can be utilized (proteins directly or via the use ofantibody agonists) to inhibit the immune response and thus ameliorateimmune related disease.

The following tests were performed:

Hematology Analysis:

Test Description: Blood tests are carried out by Abbott's Cell-Dyn3500R, an automated hematology analyzer. Some of its features include afive-part WBC differential. ‘Patient’ reports can cover over 22parameters in all.

Results:

Hematology: The (−/−) mice exhibited increased total white blood celland absolute monocyte counts when compared with those of their (+/+)littermates and the historical means. In addition, the (−/−) miceexhibited a decreased mean red blood cell count, hemoglobinconcentration, and hematocrit and an increased mean red celldistribution width. The (−/−) mice also exhibited a decreased meanplatelet count and an increased mean platelet volume.

The increased total white blood cell and absolute monocyte counts areconsistent with the pathological findings discussed above.

The (−/−) mice exhibited a decreased mean total red blood cell count,hemoglobin level, and hematocrit when compared with their (+/+)littermates and the historical means.

These results are related to a phenotype associated with anemia. Thus,PRO1380 polypeptides, agonists thereof or the encoding gene for PRO1380polypeptides must be essential for normal red blood cell production andas such would be useful in the treatment of blood disorders associatedwith anemia or a low hematocrit.

The (−/−) mice also exhibited a decreased mean platelet count whencompared with their (+/+) littermates and the historical mean.

Thus, mutant mice deficient in the DNA60740-1615 gene resulted in aphenotype related to coagulation disorders. In this regard, PRO1380polypeptides or agonists thereof would be useful in treating disordersrelated to abnormal blood coagulation such as hemophilia.

Acute Phase Response:

Test Description: Bacterial lipopolysaccharide (LPS) is an endotoxin,and as such is a potent inducer of an acute phase response and systemicinflammation. The Level I LPS mice were injected intraperitoneally(i.p.) with a sub lethal dose of LPS in 200 μL sterile saline using a 26gauge needle. The doses were based on the average weight of the micetested at 1 μg/g body weight 3 hours after injection; a 100 ul bloodsample was then taken and analyzed for the presence of TNFa, MCP-1, andIL-6 on the FACS Calibur instrument.

Results:

Acute Phase Response: The (−/−) mice exhibited increased mean serumIL-6, TNFalpha and MCP-1 responses to LPS challenge when compared withthose of their (+/+) littermates and the historical means.

Ovalbumin Challenge

Procedure: This assay was carried out on 7 wild types and 8 homozygotes.Chicken ovalbumin (OVA) is a T-cell dependent antigen, which is commonlyused as a model protein for studying antigen-specific immune responsesin mice. OVA is non-toxic and inert and therefore will not cause harm tothe animals even if no immune response is induced. The murine immuneresponse to OVA has been well characterized, to the extent that theimmunodominant peptides for eliciting T cell responses have beenidentified. Anti-OVA antibodies are detectable 8 to 10 days afterimmunization using enzyme-linked immunosorbent assay (ELIZA), anddetermination of different isotypes of antibodies gives furtherinformation on the complex processes that may lead to a deficientresponse in genetically engineered mice.

As noted above, this protocol assesses the ability of mice to raise anantigen-specific immune response. Animals were injected IP with 50 mg ofchicken ovalbumin emulsified in Complete Freund's Adjuvant and 14 dayslater the serum titer of anti-ovalbumin antibodies (IgM, IgG1 and IgG2subclasses) was measured. The amount of OVA-specific antibody in theserum sample is proportional to the Optical Density (OD) value generatedby an instrument that scans a 96-well sample plate. Data was collectedfor a set of serial dilutions of each serum sample.

Results of this Challenge:

Ovalbumin: The (−/−) mice exhibited decreased mean serum IgG1 responseto ovalbumin challenge when compared with those of their gender-matched(+/+) littermates and the historical means.

In summary, the ovalbumin challenge studies indicate that knockout micedeficient in the gene encoding PRO1380 polypeptides exhibitimmunological abnormalities when compared with their wild-typelittermates. In particular, the mutant mice exhibited a decreasedability to elicit an immunological response when challenged with theT-cell dependent OVA antigen. Accordingly, inhibitors or antagonists ofPRO1380 polypeptides would mimic these immunological findings. Theseresults are consistent with the pathology report indicating lymphoiddepletion (T cell) in the spleen, lymph nodes and thymus.

Serum Immunoglobulin Isotyping Assay:

The Serum Immunoglobulin Isotyping Assay was performed using aCytometric Bead Array (CBA) kit. This assay was used to rapidly identifythe heavy and light chain isotypes of a mouse monoclonal antibody in asingle sample. The values expressed are “relative fluorescence units”and are based on the detection of kappa light chains. Any value <6 isnot significant.

Results:

Serum Imm. 2: The (−/−) mice exhibited an increased mean serum IgA leveland IgM when compared with that of their (+/+) littermates, the (+/+)median for the project, and the cumulative (+/+) historical medians.

Mutant (−/−) mice exhibited elevation of IgA serum immunoglobulinscompared to their gender-matched (+/+) littermates. IgA mainly functionsas an epithelial cell protector which can neutralize bacterial toxinsand viruses. Although no obvious disease susceptibility is associatedwith selective IgA defects, they are commoner in people with chroniclung disease than in the general population. This suggests that lack ofIgA may result in a predisposition to lung infections with variouspathogens and is consistent with the role of IgA in defense at the bodysurfaces. In this case, the phenotype observed for knockout miceresulted in an increase in IgA serum levels suggesting that inhibitors(antagonists) of PRO1380 polypeptides would mimic these immunologicaleffects.

Fluorescence-Activated Cell-Sorting (FACS) Analysis

Procedure:

FACS analysis of immune cell composition from peripheral blood wasperformed including CD4, CD8 and T cell receptor to evaluate Tlymphocytes, CD19 for B lymphocytes, CD45 as a leukocyte marker and panNK for natural killer cells. The FACS analysis was carried out on 2 wildtype and 6 homozygous mice and included cells derived from thymus,spleen, bone marrow and lymph node.

In these studies, analyzed cells were isolated from thymus, peripheralblood, spleen, bone marrow and lymph nodes. Flow cytometry was designedto determine the relative proportions of CD4 and CD8 positive T cells, Bcells, NK cells and monocytes in the mononuclear cell population. ABecton-Dickinson FACS Calibur 3-laser FACS machine was used to assessimmune status. For Phenotypic Assays and Screening, this machine recordsCD4+/CD8−, CD8+/CD4−, NK, B cell and monocyte numbers in addition to theCD4+/CD8+ ratio.

The mononuclear cell profile was derived by staining a single sample oflysed peripheral blood from each mouse with a panel of sixlineage-specific antibodies: CD45 PerCP, anti-TCRb APC, CD4 PE, CD8FITC, pan-NK PE, and CD19 FITC. The two FITC and PE labeled antibodiesstain mutually exclusive cell types. The samples were analyzed using aBecton Dickinson FACS Calibur flow cytometer with CellQuest software.

Results:

Tissue Specific FACS Overall Observations: The (−/−) mice exhibited analtered distribution of leukocyte subsets in the peripheral blood,characterized by a decreased mean percentage of CD4 and CD8 cells inlymph nodes and spleen (approximately ⅓ of the wild-type); increasedmemory T cells (approximately 5-fold—increase in CD62LloCD44hi cells (%CD4 or CD8 total); decreased naive T cells (approximately 2-fold);increased CD25+ cells: increased thymic DN, decreased DP T cells; and anincreased mean percentage of monocytes and DC in spleen (CD11b+,CD11b+c+ (approximately 5-fold) and peritoneal lavage (CD11b+);increased CD19+ cells in LN; increased CD117 in bone marrow cells whencompared with those of their (+/+) littermates and the historical mean.

These results are consistent with the pathological observations whereinthe mutant (−/−) mice exhibited lymphoid depletion (T cells).histiocytosis, lymphadenopathy and splenomegaly.

(d) Bone Metabolism & Body Diagnostics: Radiology Phenotypic Analysis

In the area of bone metabolism, targets were identified herein for thetreatment of arthritis, osteoporosis, osteopenia and osteopetrosis aswell as identifying targets that promote bone healing. Tests included:

DEXA for measurement of bone mineral density on femur and vertebra

MicroCT for very high resolution and very high sensitivity measurementsof bone mineral density for both trabecular and cortical bone.

Dexa Analysis—Test Description:

Procedure: A cohort of 4 wild type, 4 heterozygous and 8 homozygous micewere tested in this assay. Dual Energy X-ray Absorptiometry (DEXA) hasbeen used successfully to identify changes in bone. Anesthetized animalswere examined and bone mineral content (BMC), BMC/LBM ratios, volumetricbone mineral density (vBMD), total body BMD, femur BMD and vertebra BMDwere measured.

The mouse was anesthetized by intraperitoneal injection of Avertin(1.25% 2,2,2,-tribromoethanol, 20 ml/kg body weight), body length andweight were measured, and then the mouse was placed in a prone positionon the platform of the PIXImus™ Densitometer (Lunar Inc.) for a DEXAscan. Using Lunar PIXImus software, the bone mineral density (BMD) andfat composition (% fat) and total tissue mass (TTM) were determined inthe regions of interest (ROI) [i.e., whole body, vertebrae, and bothfemurs].

CAT-Scan Protocol:

Mice were injected with a CT contrast agent, Omnipaque 300 (NycomedAmershan, 300 mg of iodine per ml, 0.25 ml per animal, or 2.50-3.75 giodine/kg of body weight) intraperitoneally. After resting in the cagefor ˜10 minutes, the mouse was then sedated by intraperitoneal injectionof Avertin (1.25% 2,2,2,-tribromoethanol, 20 ml/kg body weight). ACAT-scan was performed using a MicroCAT scanner (ImTek, Inc.) with theanesthetized animal lying prone on the test bed. Three dimensionalimages were reconstructed by the Feldkamp algorithm in a cluster ofworkstations using an ImTek 3D RECON software.

Results:

DEXA: Female (−/−) mice exhibited decreased mean percent total body fatand fat mass (g) when compared with that of their gender-matched (+/+)littermates and the historical means.

Mutant (−/−) mice deficient in the gene encoding PRO1380 polypeptidesshow a phenotype consistent with tissue wasting diseases (decreasedtotal body fat (% and g)). Thus, antagonists or inhibitors of PRO1380polypeptides or its encoding gene would mimic these metabolic and growthrelated effects. On the other hand, PRO1380 polypeptides or agoniststhereof would be useful in the prevention and/or treatment of metabolicdisorders related to abnormal fat metabolism or other tissue wastingdiseases.

CATScan: All 3 (−/−) mice (M-95, M-103, and F-129) exhibited moderatesplenomegaly.

(e) Phenotypic Analysis: Cardiology

In the area of cardiovascular biology, targets were identified hereinfor the treatment of hypertension, atherosclerosis, heart failure,stroke, various coronary artery diseases, dyslipidemias such as highcholesterol (hypercholesterolemia) and elevated serum triglycerides(hypertriglyceridemia), diabetes and/or obesity. The phenotypic testsincluded the measurement of serum cholesterol and triglycerides.

Blood Lipids

Procedure: A cohort of 4 wild type, 4 heterozygous and 8 homozygous micewere tested in this assay. High cholesterol levels and increasedtriglyceride blood levels are recognized risk factors in the developmentof cardiovascular disease and/or diabetes. Measuring blood lipidsfacilitates the finding of biological switches that regulate blood lipidlevels. Inhibition of factors which elevate blood lipid levels may beuseful for reducing the risk for cardiovascular disease. In these bloodchemistry tests, cholesterol measurements were recorded using the COBASIntegra 400 (mfr: Roche).

Results:

Blood Chemistry: Both the male and female (−/−) mice exhibited decreasedmean serum cholesterol levels when compared with those of theirgender-matched (+/+) littermates and the historical means.

In summary, these knockout mutant mice exhibited a positive phenotypewith regards to lipid metabolism. Thus, mutant mice deficient in thePRO1380 gene can serve as a model for treatment of cardiovasculardisease associated with dyslipidemia, hypertension, atherosclerosis,heart failure, stroke, or various coronary artery diseases.

(f) Phenotypic Analysis: Metabolism—Blood Chemistry

In the area of metabolism, targets may be identified for the treatmentof diabetes. Blood chemistry phenotypic analysis includes blood glucosemeasurements. The COBAS Integra 400 (mfr: Roche) was used for runningblood chemistry tests on the mice. In the area of metabolism, targetsmay be identified for the treatment of diabetes.

Results:

Both the male and female (−/−) mice exhibited decreased mean serumglucose levels when compared with those of their gender-matched (+/+)littermates and the historical means. These results may indicate anincreased insulin sensitivity in the mutant (−/−) mice.

(g) Phenotypic Analysis: CNS/Neurology

In the area of neurology, analysis focused herein on identifying in vivovalidated targets for the treatment of neurological and psychiatricdisorders including depression, generalized anxiety disorders, attentiondeficit hyperactivity disorder, obsessive compulsive disorder,schizophrenia, cognitive disorders, hyperalgesia and sensory disorders.Neurological disorders include the category defined as “anxietydisorders” which include but are not limited to: mild to moderateanxiety, anxiety disorder due to a general medical condition, anxietydisorder not otherwise specified, generalized anxiety disorder, panicattack, panic disorder with agoraphobia, panic disorder withoutagoraphobia, posttraumatic stress disorder, social phobia, specificphobia, substance-induced anxiety disorder, acute alcohol withdrawal,obsessive compulsive disorder, agoraphobia, bipolar disorder I or II,bipolar disorder not otherwise specified, cyclothymic disorder,depressive disorder, major depressive disorder, mood disorder,substance-induced mood disorder. In addition, anxiety disorders mayapply to personality disorders including but not limited to thefollowing types: paranoid, antisocial, avoidant behavior, borderlinepersonality disorders, dependent, histronic, narcissistic,obsessive-compulsive, schizoid, and schizotypal.

Procedure:

Behavioral screens were performed on a cohort of 4 wild type, 4heterozygous and 8 homozygous mutant mice. All behavioral tests weredone between 12 and 16 weeks of age unless reduced viabilitynecessitates earlier testing. These tests included open field to measureanxiety, activity levels and exploration.

Functional Observational Battery (FOB) Test—Stress-Induced Hyperthermia:

The FOB is a series of situations applied to the animal to determinegross sensory and motor deficits. A subset of tests from the Irwinneurological screen that evaluates gross neurological function is used.In general, short-duration, tactile, olfactory, and visual stimuli areapplied to the animal to determine their ability to detect and respondnormally. These simple tests take approximately 10 minutes and the mouseis returned to its home cage at the end of testing.

Results:

Anxiety: The male (−/−) mice exhibited a decreased response tostress-induced hyperthermia when compared with their gender-matched(+/+) littermates and the historical mean, suggesting a decreasedanxiety-like response in the mutants. Thus, knockout mice demonstrated aphenotype consistent with depression, generalized anxiety disorders,cognitive disorders, hyperalgesia and sensory disorders and/or bipolardisorders. Thus, PRO1380 polypeptides and agonists thereof would beuseful for the treatment or amelioration of the symptoms associated withdepressive disorders.48.19. Generation and Analysis of Mice Comprising DNA68872-1620 (UNQ722)Gene Disruptions

In these knockout experiments, the gene encoding PRO1387 polypeptides(designated as DNA68872-1620) (UNQ722) was disrupted. The gene specificinformation for these studies is as follows: the mutated mouse genecorresponds to nucleotide reference: NM_(—)001005421 Mus musculus genemodel 638, (NCBI) (Gm638); protein reference: Q80UL9 ACCESSION: Q80UL9NID: Mus musculus (Mouse). Adhesion molecule AMICA; the human genesequence reference: NM_(—)153206 ACCESSION: NM_(—)153206NID: gi 23397450refNM_(—)153206.1 Homo sapiens adhesion molecule AMICA (AMICA); thehuman protein sequence corresponds to reference: Q8N917 ACCESSION:Q8N917 NID: Homo sapiens (Human). Hypothetical protein FLJ37080.

The mouse gene of interest is AMICA (adhesion molecule AMICA), orthologof human AMICA1 (adhesion molecule, interacts with CXADR antigen 1).Aliases include GM638, gene model 638, MGC61025, JAML, FLJ37080,junctional adhesion molecule, and adhesion molecule that interacts withCXADR antigen 1.

AMICA1 is a type I integral plasma membrane protein that likelyfunctions as a cell adhesion molecule. The protein contains a signalpeptide, two extracellular immunoglobulin-like domains, a transmembranesegment, and a cytoplasmic tail. AMICA 1 is expressed primarily ingranulocytes and other hematopoietic cells and is likely to play a rolein leukocyte transmigration (Moog-Lutz et al, Blood 102(9):3371-8(2003)).

Targeted or gene trap mutations are generated in strain129SvEv^(Brd)-derived embryonic stem (ES) cells. The chimeric mice arebred to C57BL/6J albino mice to generate F1 heterozygous animals. Theseprogeny are intercrossed to generate F2 wild type, heterozygous, andhomozygous mutant progeny. On rare occasions, for example when very fewF1 mice are obtained from the chimera, F1 heterozygous mice are crossedto 129SvEv^(Brd)/C57 hybrid mice to yield additional heterozygousanimals for the intercross to generate the F2 mice. Level I phenotypicanalysis is performed on mice from this generation

wt het hom Total Observed 20 40 12 72 Expected 18 36 18 72 Chi-Sq. =0.09 Significance = 0.95599747 (hom/n) = 0.24 Avg. Litter Size = 8Mutation InformationMutation Type Homologous Recombination (standard)Description: Coding exons 1 through 3 were targeted (NCBI accessionNM_(—)001005421.2).1. Wild-type Expression Panel: Expression of the target gene wasdetected in embryonic stem (ES) cells and in all 13 adult tissue samplestested by RT-PCR, except skin fibroblast.2. QC Expression: QC Images: Disruption of the target gene was confirmedby Southern hybridization analysis.

48.19.1. Phenotypic Analysis (for Disrupted Gene: DNA68872-1620 (UNQ722)

(a) Overall Phenotypic Summary:

Mutation of the gene encoding the ortholog of human adhesion molecule,interacts with CXADR antigen 1 (AMICA1) resulted in a decreased meanserum IgG2a response to ovalbumin challenge in (−/−) mice. Homozygous(−/−) mice also exhibited a decreased latency to respond during hotplate testing. The mutant female (−/−) mice showed increased total bodyfat. Gene disruption was confirmed by Southern blot.

(b) Immunology Phenotypic Analysis

Immune related and inflammatory diseases are the manifestation orconsequence of fairly complex, often multiple interconnected biologicalpathways which in normal physiology are critical to respond to insult orinjury, initiate repair from insult or injury, and mount innate andacquired defense against foreign organisms. Disease or pathology occurswhen these normal physiological pathways cause additional insult orinjury either as directly related to the intensity of the response, as aconsequence of abnormal regulation or excessive stimulation, as areaction to self, or as a combination of these.

Though the genesis of these diseases often involves multistep pathwaysand often multiple different biological systems/pathways, interventionat critical points in one or more of these pathways can have anameliorative or therapeutic effect. Therapeutic intervention can occurby either antagonism of a detrimental process/pathway or stimulation ofa beneficial process/pathway.

T lymphocytes (T cells) are an important component of a mammalian immuneresponse. T cells recognize antigens which are associated with aself-molecule encoded by genes within the major histocompatibilitycomplex (MHC). The antigen may be displayed together with MHC moleculeson the surface of antigen presenting cells, virus infected cells, cancercells, grafts, etc. The T cell system eliminates these altered cellswhich pose a health threat to the host mammal. T cells include helper Tcells and cytotoxic T cells. Helper T cells proliferate extensivelyfollowing recognition of an antigen-MHC complex on an antigen presentingcell. Helper T cells also secrete a variety of cytokines, i.e.,lymphokines, which play a central role in the activation of B cells,cytotoxic T cells and a variety of other cells which participate in theimmune response.

In many immune responses, inflammatory cells infiltrate the site ofinjury or infection. The migrating cells may be neutrophilic,eosinophilic, monocytic or lymphocytic as can be determined byhistological examination of the affected tissues. Current Protocols inImmunology, ed. John E. Coligan, 1994, John Wiley & Sons, Inc.

Many immune related diseases are known and have been extensivelystudied. Such diseases include immune-mediated inflammatory diseases(such as rheumatoid arthritis, immune mediated renal disease,hepatobiliary diseases, inflammatory bowel disease (IBD), psoriasis, andasthma), non-immune-mediated inflammatory diseases, infectious diseases,immunodeficiency diseases, neoplasia, and graft rejection, etc. In thearea of immunology, targets were identified for the treatment ofinflammation and inflammatory disorders.

In the area of immunology, targets have been identified herein for thetreatment of inflammation and inflammatory disorders. Immune relateddiseases, in one instance, could be treated by suppressing the immuneresponse. Using neutralizing antibodies that inhibit molecules havingimmune stimulatory activity would be beneficial in the treatment ofimmune-mediated and inflammatory diseases. Molecules which inhibit theimmune response can be utilized (proteins directly or via the use ofantibody agonists) to inhibit the immune response and thus ameliorateimmune related disease.

The following test was performed:

Ovalbumin Challenge

Procedure: This assay was carried out on 7 wild types and 8 homozygousmice. Chicken ovalbumin (OVA) is a T-cell dependent antigen, which iscommonly used as a model protein for studying antigen-specific immuneresponses in mice. OVA is non-toxic and inert and therefore will notcause harm to the animals even if no immune response is induced. Themurine immune response to OVA has been well characterized, to the extentthat the immunodominant peptides for eliciting T cell responses havebeen identified. Anti-OVA antibodies are detectable 8 to 10 days afterimmunization using enzyme-linked immunosorbent assay (ELIZA), anddetermination of different isotypes of antibodies gives furtherinformation on the complex processes that may lead to a deficientresponse in genetically engineered mice.

As noted above, this protocol assesses the ability of mice to raise anantigen-specific immune response. Animals were injected IP with 50 mg ofchicken ovalbumin emulsified in Complete Freund's Adjuvant and 14 dayslater the serum titer of anti-ovalbumin antibodies (IgM, IgG1 and IgG2subclasses) was measured. The amount of OVA-specific antibody in theserum sample is proportional to the Optical Density (OD) value generatedby an instrument that scans a 96-well sample plate. Data was collectedfor a set of serial dilutions of each serum sample.

Results of this Challenge:

The (−/−) mice exhibited decreased mean serum IgG2a response whencompared with their (+/+) littermates and the historical mean.

In summary, the ovalbumin challenge studies indicate that knockout micedeficient in the gene encoding PRO1387 polypeptides exhibitimmunological abnormalities when compared with their wild-typelittermates. In particular, the mutant mice exhibited a decreasedability to elicit an immunological response when challenged with theT-cell dependent OVA antigen. Thus, PRO1387 polypeptides or agoniststhereof, would be useful for stimulating the immune system (such as Tcell proliferation) and would find utility in the cases wherein thiseffect would be beneficial to the individual such as in the case ofleukemia, and other types of cancer, and in immuno-compromised patients,such as AIDS sufferers. Accordingly, inhibitors (antagonists) of PRO1387polypeptides would be useful for inhibiting the immune response and thuswould be useful candidates for suppressing harmful immune responses,e.g. in the case of graft rejection or graft-versus-host diseases.

(c) Bone Metabolism & Body Diagnostics: Radiology Phenotypic Analysis

In the area of bone metabolism, targets were identified herein for thetreatment of arthritis, osteoporosis, osteopenia and osteopetrosis aswell as identifying targets that promote bone healing. Tests included:

DEXA for measurement of bone mineral density on femur and vertebra

MicroCT for very high resolution and very high sensitivity measurementsof bone mineral density for both trabecular and cortical bone.

Dexa Analysis—Test Description:

Procedure: A cohort of 4 wild type, 4 heterozygous and 8 homozygous micewere tested in this assay. Dual Energy X-ray Absorptiometry (DEXA) hasbeen used successfully to identify changes in bone. Anesthetized animalswere examined and bone mineral content (BMC), BMC/LBM ratios, volumetricbone mineral density (vBMD), total body BMD, femur BMD and vertebra BMDwere measured.

The mouse was anesthetized by intraperitoneal injection of Avertin(1.25% 2,2,2,-tribromoethanol, 20 ml/kg body weight), body length andweight were measured, and then the mouse was placed in a prone positionon the platform of the PIXImus™ Densitometer (Lunar Inc.) for a DEXAscan. Using Lunar PIXImus software, the bone mineral density (BMD) andfat composition (% fat) and total tissue mass (TTM) were determined inthe regions of interest (ROI) [i.e., whole body, vertebrae, and bothfemurs].

Results:

The female (−/−) mice exhibited increased total body fat (% and g) whencompared to their gender matched wild-type (+/+) littermates andhistorical mean.

These studies suggest that mutant (−/−) non-human transgenic animalsexhibit a negative phenotype that would be associated with obesity.Thus, PRO1387 polypeptides or agonists thereof are essential for normalgrowth and metabolic processes and especially would be important in theprevention and/or treatment of obesity.

(d) Phenotypic Analysis: CNS/Neurology

In the area of neurology, analysis focused herein on identifying in vivovalidated targets for the treatment of neurological and psychiatricdisorders including depression, generalized anxiety disorders, attentiondeficit hyperactivity disorder, obsessive compulsive disorder,schizophrenia, cognitive disorders, hyperalgesia and sensory disorders.Neurological disorders include the category defined as “anxietydisorders” which include but are not limited to: mild to moderateanxiety, anxiety disorder due to a general medical condition, anxietydisorder not otherwise specified, generalized anxiety disorder, panicattack, panic disorder with agoraphobia, panic disorder withoutagoraphobia, posttraumatic stress disorder, social phobia, specificphobia, substance-induced anxiety disorder, acute alcohol withdrawal,obsessive compulsive disorder, agoraphobia, bipolar disorder I or II,bipolar disorder not otherwise specified, cyclothymic disorder,depressive disorder, major depressive disorder, mood disorder,substance-induced mood disorder. In addition, anxiety disorders mayapply to personality disorders including but not limited to thefollowing types: paranoid, antisocial, avoidant behavior, borderlinepersonality disorders, dependent, histronic, narcissistic,obsessive-compulsive, schizoid, and schizotypal.

Procedure:

Behavioral screens were performed on a cohort of 4 wild type, 4heterozygous and 8 homozygous mutant mice. All behavioral tests weredone between 12 and 16 weeks of age unless reduced viabilitynecessitates earlier testing. These tests included open field to measureanxiety, activity levels and exploration.

Functional Observational Battery (FOB) Test—Hot Plate Testing:

The FOB is a series of situations applied to the animal to determinegross sensory and motor deficits. A subset of tests from the Irwinneurological screen that evaluates gross neurological function is used.In general, short-duration, tactile, olfactory, and visual stimuli areapplied to the animal to determine their ability to detect and respondnormally. These simple tests take approximately 10 minutes and the mouseis returned to its home cage at the end of testing.

Hot Plate Testing

Test Description: The hot plate test for nociception is carried out byplacing each mouse on a small enclosed 55° C. hot plate. Latency to ahind limb response (lick, shake, or jump) is recorded, with a maximumtime on the hot plate of 30 sec. Each animal is tested once.

Results:

The (−/−) mice exhibited a decreased latency in hot plate testing whichindicates an increased pain perception compared with theirgender-matched wild-type littermates and the historical means.

48.20. Generation and Analysis of Mice Comprising DNA71290-1630 (UNQ733)Gene Disruptions

In these knockout experiments, the gene encoding PRO1419 polypeptides(designated as DNA71290-1630) (UNQ733) was disrupted. The gene specificinformation for these studies is as follows: the mutated mouse genecorresponds to nucleotide reference: BC037156 Mus musculus cDNA sequenceBC037156; the human gene sequence reference: NM_(—)152997 Homo sapienschromosome 4 open reading frame 7 (C4orf7); the human protein sequencecorresponds to reference: Q8NFU4 ACCESSION: Q8NFU4 NID: Homo sapiens(Human). Follicular dendritic cell secreted peptide precursor (FDC-SP)(FDC secreted protein).

The mouse gene of interest is cDNA sequence BC037156, ortholog of humanC4orf7 (chromosome 4 open reading frame 7). Aliases include MGC71894,FDC-SP, FDCSP, and follicular dendritic cell secreted peptide.

C4orf7 is an 84-amino acid secreted protein expressed primarily byfollicular dendritic cells of tonsils that likely functions as asignal-transducing ligand. C4orf7 expression can be stimulated by tumornecrosis factor-alpha in follicular dendritic cells and bylipopolysaccharides in blood leukocytes. Moreover, C4orf7 is expressedat very high levels in inflamed tonsillar crypts. In addition totonsils, C4orf7 is also expressed in a number of other tissues,including lymph node, trachea, prostate, thyroid, stomach, colon,spleen, peripheral blood leukocytes, and bone marrow. The protein iscapable of binding to activated B-cells, suggesting that C4orf7 plays arole in immune function (Marshall et al, J Immunol 169(5):2381-9(2002)).

Targeted or gene trap mutations are generated in strain129SvEv^(Brd)-derived embryonic stem (ES) cells. The chimeric mice arebred to C57BL/6J albino mice to generate F1 heterozygous animals. Theseprogeny are intercrossed to generate F2 wild type, heterozygous, andhomozygous mutant progeny. On rare occasions, for example when very fewF1 mice are obtained from the chimera, F1 heterozygous mice are crossedto 129SvEv^(Brd)/C57 hybrid mice to yield additional heterozygousanimals for the intercross to generate the F2 mice. Level I phenotypicanalysis is performed on mice from this generation

wt het hom Total Observed 26 43 17 86 Expected 21.5 43 21.5 86 Chi-Sq. =0.81 Significance = 0.6669768 (hom/n) = 0.23 Avg. Litter Size = 10Mutation InformationMutation Type Homologous Recombination (standard)Description: Coding exons 1 and 2 were targeted (NCBI accessionBC037156).1. Wild-type Expression Panel: Expression of the target gene wasdetected only in eye and thymus among the 13 adult tissue samples testedby RT-PCR.2. QC Expression: QC Images: Disruption of the target gene was confirmedby Southern hybridization analysis.

48.20.1. Phenotypic Analysis (for Disrupted Gene: DNA71290-1630 (UNQ733)

(a) Overall Phenotypic Summary:

Mutation of the gene encoding the ortholog of human chromosome 4 openreading frame 7 (C4orf7) resulted in the mutant (−/−) mice exhibiting anincrease in IgG2a response to the ovalbumin challenge. Gene disruptionwas confirmed by Southern blot.

(b) Immunology Phenotypic Analysis

Immune related and inflammatory diseases are the manifestation orconsequence of fairly complex, often multiple interconnected biologicalpathways which in normal physiology are critical to respond to insult orinjury, initiate repair from insult or injury, and mount innate andacquired defense against foreign organisms. Disease or pathology occurswhen these normal physiological pathways cause additional insult orinjury either as directly related to the intensity of the response, as aconsequence of abnormal regulation or excessive stimulation, as areaction to self, or as a combination of these.

Though the genesis of these diseases often involves multistep pathwaysand often multiple different biological systems/pathways, interventionat critical points in one or more of these pathways can have anameliorative or therapeutic effect. Therapeutic intervention can occurby either antagonism of a detrimental process/pathway or stimulation ofa beneficial process/pathway.

T lymphocytes (T cells) are an important component of a mammalian immuneresponse. T cells recognize antigens which are associated with aself-molecule encoded by genes within the major histocompatibilitycomplex (MHC). The antigen may be displayed together with MHC moleculeson the surface of antigen presenting cells, virus infected cells, cancercells, grafts, etc. The T cell system eliminates these altered cellswhich pose a health threat to the host mammal. T cells include helper Tcells and cytotoxic T cells. Helper T cells proliferate extensivelyfollowing recognition of an antigen-MHC complex on an antigen presentingcell. Helper T cells also secrete a variety of cytokines, i.e.,lymphokines, which play a central role in the activation of B cells,cytotoxic T cells and a variety of other cells which participate in theimmune response.

In many immune responses, inflammatory cells infiltrate the site ofinjury or infection. The migrating cells may be neutrophilic,eosinophilic, monocytic or lymphocytic as can be determined byhistological examination of the affected tissues. Current Protocols inImmunology, ed. John E. Coligan, 1994, John Wiley & Sons, Inc.

Many immune related diseases are known and have been extensivelystudied. Such diseases include immune-mediated inflammatory diseases(such as rheumatoid arthritis, immune mediated renal disease,hepatobiliary diseases, inflammatory bowel disease (IBD), psoriasis, andasthma), non-immune-mediated inflammatory diseases, infectious diseases,immunodeficiency diseases, neoplasia, and graft rejection, etc. In thearea of immunology, targets were identified for the treatment ofinflammation and inflammatory disorders.

In the area of immunology, targets have been identified herein for thetreatment of inflammation and inflammatory disorders. Immune relateddiseases, in one instance, could be treated by suppressing the immuneresponse. Using neutralizing antibodies that inhibit molecules havingimmune stimulatory activity would be beneficial in the treatment ofimmune-mediated and inflammatory diseases. Molecules which inhibit theimmune response can be utilized (proteins directly or via the use ofantibody agonists) to inhibit the immune response and thus ameliorateimmune related disease.

The following test was performed:

Ovalbumin Challenge

Procedure: This assay was carried out on 7 wild types and 8 homozygousmice. Chicken ovalbumin (OVA) is a T-cell dependent antigen, which iscommonly used as a model protein for studying antigen-specific immuneresponses in mice. OVA is non-toxic and inert and therefore will notcause harm to the animals even if no immune response is induced. Themurine immune response to OVA has been well characterized, to the extentthat the immuno-dominant peptides for eliciting T cell responses havebeen identified. Anti-OVA antibodies are detectable 8 to 10 days afterimmunization using enzyme-linked immunosorbent assay (ELIZA), anddetermination of different isotypes of antibodies gives furtherinformation on the complex processes that may lead to a deficientresponse in genetically engineered mice.

As noted above, this protocol assesses the ability of mice to raise anantigen-specific immune response. Animals were injected IP with 50 mg ofchicken ovalbumin emulsified in Complete Freund's Adjuvant and 14 dayslater the serum titer of anti-ovalbumin antibodies (IgM, IgG1 and IgG2subclasses) was measured. The amount of OVA-specific antibody in theserum sample is proportional to the Optical Density (OD) value generatedby an instrument that scans a 96-well sample plate. Data was collectedfor a set of serial dilutions of each serum sample.

Results of this Challenge:

The (−/−) mice exhibited an increased mean serum IgG2a response whencompared with their (+/+) littermates and the historical mean.

In summary, the ovalbumin challenge studies indicate that knockout micedeficient in the gene encoding PRO1419 polypeptides exhibitimmunological abnormalities when compared with their wild-typelittermates. UNQ733 may play a role in inflammatory disorders sinceUNQ733 is secreted by follicular dendritic cells and it specificallybinds B cells (which can be induced by TNF alpha). Hyperplasia ofadenoid and tonsils have also been observed. In particular, the mutantmice exhibited a decreased ability to elicit an immunological responsewhen challenged with the T-cell dependent OVA antigen. Thus, inhibitors(antagonists) of PRO1419 polypeptides would be useful for stimulatingthe immune system (such as T cell proliferation) and would find utilityin the cases wherein this effect would be beneficial to the individualsuch as in the case of leukemia, and other types of cancer, and inimmuno-compromised patients, such as AIDS sufferers. Accordingly,PRO1419 polypeptides or agonists thereof would be useful for inhibitingthe immune response and thus would be useful candidates for suppressingharmful immune responses, e.g. in the case of graft rejection orgraft-versus-host diseases.

48.21. Generation and Analysis of Mice Comprising DNA71184-1634 (UNQ738)Gene Disruptions

In these knockout experiments, the gene encoding PRO1433 polypeptides(designated as DNA71184-1634) (UNQ738) was disrupted. The gene specificinformation for these studies is as follows: the mutated mouse genecorresponds to nucleotide reference: NM_(—)026384 Mus musculusdiacylglycerol O-acyltransferase 2 (Dgat2); protein reference: Q9DCV3ACCESSION: Q9DCV3 NID: Mus musculus (Mouse). 0610010B06Rik protein(Diacylglycerol acyltransferase 2); the human gene sequence reference:NM_(—)032564 ACCESSION: NM_(—)032564 NID: gi 26024196 ref NM_(—)032564.2Homo sapiens diacylglycerol O-acyltransferase homolog 2 (mouse) (DGAT2);the human protein sequence corresponds to reference: Q96PD7 ACCESSION:Q96PD7 NID: Homo sapiens (Human). Diacylglycerol acyltransferase 2(Hypothetical protein) (GS1999full protein).

The mouse gene of interest is Dgat2 (diacylglycerol O-acyltransferase2), ortholog of human DGAT2 (diacylglycerol O-acyltransferase homolog 2[mouse]). Aliases include DGAT-2, 0610010B06Rik, diacylglycerolacyltransferase 2, HMFN1045, and GS1999full.

DGAT2 is an integral membrane protein located on the endoplasmicreticulum that functions as an enzyme, catalyzing the formation oftriglycerides from long-chain acyl-CoAs and diacylglycerol. Expressionof DGAT2 is ubiquitous but seems most abundant in liver, white fat,mammary gland, small intestine, and sebaceous glands of skin. DGAT2plays a role in energy metabolism and skin barrier function (Cases etal, J Biol Chem 276(42):38870-6 (2001); Wakimoto et al, Biochem BiophysRes Commun 310(2):296-302 (2003); Stone et al, Biol Chem279(12):11767-76 (2004)). Moreover, DGAT2 may be associated withdiabetes and psoriasis (Watermann and Zammit, Int J Obes Relat MetabDisord 26(5):742-3 (2002); Meegalla et al, Biochem Biophys Res Commun298(3):317-23 (2002); Wakimoto et al, Biochem Biophys Res Commun310(2):296-302 (2003)).

Scot Stone and colleagues (2004) investigated the physiological role ofDGAT2 using knockout mice. They showed that DGAT2 (−/−) mice werelipopenic and died early after birth. Tissue triglyceride and energysubstrate content was severely lower in DGAT2 (−/−) mice than in (+/+)mice. Moreover, skin abnormalities and barrier maintenance function wereevident in DGAT2 (−/−) mice but not (+/+) mice, suggesting that energyhomeostasis abnormalities and dehydration contributed to early postnatallethality of the DGAT2 (−/−) mice. Scot and colleagues concluded thatthe majority of triglyceride biosynthesis in mice involves DGAT2 andthat inhibition of DGAT2 for therapeutic purposes should be approachedwith caution because DGAT2 function appears to be crucial for survival.

Targeted or gene trap mutations are generated in strain129SvEv^(Brd)-derived embryonic stem (ES) cells. The chimeric mice arebred to C57BL/6J albino mice to generate F1 heterozygous animals. Theseprogeny are intercrossed to generate F2 wild type, heterozygous, andhomozygous mutant progeny. On rare occasions, for example when very fewF1 mice are obtained from the chimera, F1 heterozygous mice are crossedto 129SvEv^(Brd)/C57 hybrid mice to yield additional heterozygousanimals for the intercross to generate the F2 mice. Level I phenotypicanalysis is performed on mice from this generation

wt het hom Total Observed 20 36 14 70 Expected 17.5 35 17.5 70 Chi-Sq. =2.62 Significance = 0.26982006 (hom/n) = 0.18 Avg. Litter Size = 8Mutation InformationMutation Type Homologous Recombination (standard)Description: Coding exon 1 was targeted (NCBI accession NM_(—)026384.2).1. Wild-type Expression Panel: Expression of the target gene wasdetected in all 13 adult tissue samples tested by RT-PCR, exceptadipose.2. QC Expression: QC Images: Disruption of the target gene was confirmedby Southern hybridization analysis.

48.21.1. Phenotypic Analysis (for Disrupted Gene: DNA71184-1634 (UNQ738)

(a) Overall Phenotypic Summary:

Mutation of the UNQ738 gene encoding the ortholog of humandiacylglycerol O-acyltransferase homolog 2 (mouse) (DGAT2) resulted inperinatal lethality of (−/−) mutants. Genetic data indicate that thismutation resulted in perinatal lethality of the homozygous mutants. Thehomozygous mutant mice were small and frail, dying within 5 days ofbirth. Necropsy revealed that the mutants were dehydrated with decreasedsubcutaneous fat depots (suggesting abnormal lipid metabolism) anddecreased lymphocytes in the spleen and thymus. Disruption of the targetgene was confirmed by Southern hybridization analysis.

(b) Pathology

Gross: The (−/−) mice were small, dehydrated, and exhibited decreasedsubcutaneous fat depots.

Microscopic: Perinatal mortality was noted in the (−/−) mice. The (−/−)mice exhibited decreased lymphocytes in the thymus and spleen. Externalknockout mice were reportedly lipopenic and died soon after birth,apparently from a profound reduction in substrates for energy metabolismand from impaired permeability barrier function in the skin.Gene Expression: LacZ activity was detected in testis and epididymisamong the panel of tissues analyzed by immunohistochemistry.

Discussion Related to Embryonic Developmental Abnormality of Lethality:

Embryonic lethality in knockout mice usually results from variousserious developmental problems including but not limited toneuro-degenerative diseases, angiogenic disorders, inflammatorydiseases, or where the gene/protein has an important role in basic cellsignaling processes in many cell types. In addition, embryonic lethalsare useful as potential cancer models. Likewise, the correspondingheterozygous (+/−) mutant animals are particularly useful when theyexhibit a phenotype and/or a pathology report which reveals highlyinformative clues as to the function of the knocked-out gene. Forinstance, EPO knockout animals were embryonic lethals, but the pathologyreports on the embryos showed a profound lack of RBCs.

(c) Bone Metabolism & Body Diagnostics: Radiology Phenotypic Analysis

In the area of bone metabolism, targets were identified herein for thetreatment of arthritis, osteoporosis, osteopenia and osteopetrosis aswell as identifying targets that promote bone healing. Tests included:

DEXA for measurement of bone mineral density on femur and vertebra

MicroCT for very high resolution and very high sensitivity measurementsof bone mineral density for both trabecular and cortical bone.

Dexa Analysis—Test Description:

Procedure: A cohort of 4 wild type, and 4 heterozygous mice were testedin this assay. Dual Energy X-ray Absorptiometry (DEXA) has been usedsuccessfully to identify changes in bone. Anesthetized animals wereexamined and bone mineral content (BMC), BMC/LBM ratios, volumetric bonemineral density (vBMD), total body BMD, femur BMD and vertebra BMD weremeasured.

The mouse was anesthetized by intraperitoneal injection of Avertin(1.25% 2,2,2,-tribromoethanol, 20 ml/kg body weight), body length andweight were measured, and then the mouse was placed in a prone positionon the platform of the PIXImus™ Densitometer (Lunar Inc.) for a DEXAscan. Using Lunar PIXImus software, the bone mineral density (BMD) andfat composition (% fat) and total tissue mass (TTM) were determined inthe regions of interest (ROI) [i.e., whole body, vertebrae, and bothfemurs].

Bone MicroCT Analysis:

Procedure: MicroCT was also used to get very sensitive measurements ofBMD. One vertebra and 1 femur were taken from a cohort of 4 wild typeand 4 heterozygous mice. Measurements were taken of lumbar 5 vertebratrabecular bone volume, trabecular thickness, connectivity density andmidshaft femur total bone area and cortical thickness. The μCT40 scansprovided detailed information on bone mass and architecture. Multiplebones were placed into sample holders and scanned automatically.Instrument software was used to select regions of interest for analysis.Trabecular bone parameters were analyzed in the fifth lumbar vertebrae(LV5) at 16 micrometer resolution and cortical bone parameters wereanalyzed in the femur midshaft at a resolution of 20 micrometers.

Results:

Micro CT: Male heterozygous (+/−) mice exhibited increased trabecularnumber and connectivity density when compared with their gender-matchedwild-type littermates and the historical mean.

In summary, the (+/−) mice exhibited increased trabecular number andconnectivity density when compared with their gender-matched (+/+)littermates. These results indicate that the knockout mutant phenotypemay be associated with such bone abnormalities as osteopetrosis.Osteopetrosis is a condition characterized by abnormal thickening andhardening of bone and abnormal fragility of the bones. As such, PRO1433polypeptides or agonists thereof would be beneficial for the treatmentof osteopetrosis or other osteo-related diseases.

48.22. Generation and Analysis of Mice Comprising DNA73739-1645 (UNQ745)Gene Disruptions

In these knockout experiments, the gene encoding PRO1474 polypeptides(designated as DNA73739-1645) (UNQ745) was disrupted. The gene specificinformation for these studies is as follows: the mutated mouse genecorresponds to nucleotide reference: NM_(—)001001803 Mus musculusesophagus cancer-related gene-2 (Ecg2); protein reference: Q61E32ACCESSION: Q61E32 NID: Mus musculus (Mouse). Esophagus cancer-relatedgene-2 precursor; the human gene sequence reference: NM_(—)032566 Homosapiens esophagus cancer-related gene-2 (ECG2); the human proteinsequence corresponds to reference: P58062 Esophagus cancer-relatedgene-2 protein precursor (ECRG-2) (UNQ745/PRO1474).

The mouse gene of interest is Ecg2 (esophagus cancer-related gene-2),ortholog of human ECG2. Aliases include ECRG2.

ECG2 is a putative secreted protein that likely functions as a proteaseinhibitor (Puente and Lopez-Otin, Genome Res 14(4):609-22 (2004)). Theprotein contains a signal peptide and a Kazal-type serine proteaseinhibitor domain (SMART accession SM00280). ECG2 is also a tumorsuppressor candidate that can associate with metallothionein 2A.Ectopically expressed ECG2 in esophageal cancer cells colocalizes withmetallothionein in the nucleus and cytoplasm, inhibits cellproliferation, and induces apoptosis. Mutations in the ECG2 gene havebeen implicated in esophageal squamous cell carcinoma (Yue et al, Int JCancer 108(2):232-6 (2004); Cui et al, Biochem Biophys Res Commun302(4):904-15 (2003)).

Targeted or gene trap mutations are generated in strain129SvEv^(Brd)-derived embryonic stem (ES) cells. The chimeric mice arebred to C57BL/6J albino mice to generate F1 heterozygous animals. Theseprogeny are intercrossed to generate F2 wild type, heterozygous, andhomozygous mutant progeny. On rare occasions, for example when very fewF1 mice are obtained from the chimera, F1 heterozygous mice are crossedto 129SvEv^(Brd)/C57 hybrid mice to yield additional heterozygousanimals for the intercross to generate the F2 mice. Level I phenotypicanalysis is performed on mice from this generation

wt het hom Total Observed 16 42 22 80 Expected 20 40 20 80 Chi-Sq. =1.38 Significance = 0.50157607 (hom/n) = 0.28 Avg. Litter Size = 9Mutation InformationMutation Type: Homologous Recombination (standard)Description: Coding exons 1 through 3 were targeted (NCBI accessionNM_(—)001001803.1).1. Wild-type Expression Panel: Expression of the target gene wasdetected only in eye among the 13 adult tissue samples tested by RT-PCR.2. QC Expression: QC Images: Disruption of the target gene was confirmedby Southern hybridization analysis.

48.22.1. Phenotypic Analysis (for Disrupted Gene: DNA73739-1645 (UNQ745)

(a) Overall Phenotypic Summary:

Mutation of the gene encoding the ortholog of human esophaguscancer-related gene-2 (ECG2) resulted in the homozygous (−/−) miceexhibiting decreased total tissue mass and decreased bone mineraldensity measurements. Some of the female (−/−) mice were fat showingincreased total body fat and high triglyceride levels. Gene disruptionwas confirmed by Southern blot.

(b) Bone Metabolism & Body Diagnostics: Radiology Phenotypic Analysis

In the area of bone metabolism, targets were identified herein for thetreatment of arthritis, osteoporosis, osteopenia and osteopetrosis aswell as identifying targets that promote bone healing. Tests included:

DEXA for measurement of bone mineral density on femur and vertebra

MicroCT for very high resolution and very high sensitivity measurementsof bone mineral density for both trabecular and cortical bone.

Dexa Analysis—Test Description:

Procedure: A cohort of 4 wild type, 4 heterozygous and 8 homozygous weretested in this assay. Dual Energy X-ray Absorptiometry (DEXA) has beenused successfully to identify changes in bone. Anesthetized animals wereexamined and bone mineral content (BMC), BMC/LBM ratios, volumetric bonemineral density (vBMD), total body BMD, femur BMD and vertebra BMD weremeasured.

The mouse was anesthetized by intraperitoneal injection of Avertin(1.25% 2,2,2,-tribromoethanol, 20 ml/kg body weight), body length andweight were measured, and then the mouse was placed in a prone positionon the platform of the PIXImus™ Densitometer (Lunar Inc.) for a DEXAscan. Using Lunar PIXImus software, the bone mineral density (BMD) andfat composition (% fat) and total tissue mass (TTM) were determined inthe regions of interest (ROI) [i.e., whole body, vertebrae, and bothfemurs].

Bone MicroCT Analysis:

Procedure: MicroCT was also used to get very sensitive measurements ofBMD. One vertebra and 1 femur were taken from a cohort of 4 wild typeand 8 homozygous mice. Measurements were taken of lumbar 5 vertebratrabecular bone volume, trabecular thickness, connectivity density andmidshaft femur total bone area and cortical thickness. The μCT40 scansprovided detailed information on bone mass and architecture. Multiplebones were placed into sample holders and scanned automatically.Instrument software was used to select regions of interest for analysis.Trabecular bone parameters were analyzed in the fifth lumbar vertebrae(LV5) at 16 micrometer resolution and cortical bone parameters wereanalyzed in the femur midshaft at a resolution of 20 micrometers.

Results:

DEXA: The male (−/−) mice exhibited decreased mean total body and femurbone mineral density (BMD) as well as a decrease in total tissue masswhen compared with the those of their gender-matched (+/+) littermatesand the historical means. In addition, a few of the female (−/−) miceshowed high total body fat [One or two female (−/−) mice were fat withincreased total body fat and showed high mean serum triglyceridelevels]. Overall population did not display characteristics ofobesity/type 2 diabetes.

The (−/−) mice analyzed by DEXA analysis exhibited decreased bonemeasurements when compared with their (+/+) littermates, suggestive ofabnormal bone disorders. The (−/−) mice exhibited a negative bonephenotype with abnormal and decreased bone measurements reflective ofbone metabolic disorders. The negative bone phenotype indicates thatPRO1474 polypeptides or agonists thereof would be useful for maintainingbone homeostasis. In addition, PRO1474 polypeptides would be useful inbone healing or for the treatment of arthritis or osteoporosis, whereasantagonists (or inhibitors) of PRO1474 polypeptides or its encoding genewould lead to abnormal or pathological bone disorders includinginflammatory diseases associated with abnormal bone metabolism includingarthritis, osteoporosis and osteopenia.

48.23. Generation and Analysis of Mice Comprising DNA76393-1664 (UNQ762)Gene Disruptions

In these knockout experiments, the gene encoding PRO1550 polypeptides(designated as DNA76393-1664) (UNQ762) was disrupted. The gene specificinformation for these studies is as follows: the mutated mouse genecorresponds to nucleotide reference: AK003674 Mus musculus 18-day embryowhole body cDNA, RIKEN full-length enriched library, clone: 1110014B07product:hypothetical Collagen triple helix repeat containing protein,full insert sequence; protein reference: Q9D1D6 ACCESSION: Q9D1D6 NID:Mus musculus (Mouse). 1110014B07Rik protein; the human gene sequencereference: NM_(—)138455 ACCESSION: NM_(—)138455 NID: gi 34147546refNM_(—)138455.2 Homo sapiens collagen triple helix repeat containing 1(CTHRC1); the human protein sequence corresponds to reference: Q96CG8ACCESSION: Q96CG8 NID: Homo sapiens (Human). Similar to RIKEN cDNA1110014B07 gene (Collagen triple helix repeat-containing protein 1).

The mouse gene of interest is Cthrc1 (collagen triple helix repeatcontaining 1), ortholog of human CTHRC1. Aliases include 1110014B07Rik.

CTHRC1 is a secreted protein, containing a signal peptide and a collagentriple-helix repeat. CTHRC1 is expressed in fibroblasts of remodelingadventitia and smooth muscle cells of neointima of balloon-injuredvascular tissue and is also found in the matrix of calcifying humanatherosclerotic plaques. CTHRC1 is not expressed in normal arteries.Expression of CTHRC1 is upregulated in response to transforming growthfactor-beta and bone morphogenic protein-4. CTHRC1 inhibits expressionand secretion of collagen type I and enhances cell migration. Thus,CTHRC1 appears to play a role in vascular remodeling by inhibitingdeposition of collagen and promoting migration of vascular cells (Pyagayet al, Circ Res 96(2):261-8 (2005)).

Targeted or gene trap mutations are generated in strain129SvEv^(Brd)-derived embryonic stem (ES) cells. The chimeric mice arebred to C57BL/6J albino mice to generate F1 heterozygous animals. Theseprogeny are intercrossed to generate F2 wild type, heterozygous, andhomozygous mutant progeny. On rare occasions, for example when very fewF1 mice are obtained from the chimera, F1 heterozygous mice are crossedto 129SvEv^(Brd)/C57 hybrid mice to yield additional heterozygousanimals for the intercross to generate the F2 mice. Level I phenotypicanalysis is performed on mice from this generation

wt het hom Total Observed 16 33 17 66 Expected 16.5 33 16.5 66 Chi-Sq. =3.43 Significance = 0.17996371 (hom/n) = 0.27 Avg. Litter Size = 9Mutation InformationMutation Type: Homologous Recombination (standard)Description: Coding exons 1 and 2 were targeted (NCBI accessionAK076498).1. Wild-type Expression Panel: Expression of the target gene wasdetected in all 13 adult tissue samples tested by RT-PCR, except liverand bone.2. QC Expression: QC Images: Disruption of the target gene was confirmedby Southern hybridization analysis.

48.23.1. Phenotypic Analysis (for Disrupted Gene: DNA76393-1664 (UNQ762)

(a) Overall Phenotypic Summary:

Mutation of the gene encoding the ortholog of human collagen triplehelix repeat containing 1 (CTHRC1) resulted in increased serum alkalinephosphatase levels in both (+/−) and (−/−) mice. Female (−/−) mice alsoexhibited a notably decreased skin proliferation rate. Gene disruptionwas confirmed by Southern blot.

(b) Expression Patterns:

GeneLogic analysis shows UNQ762 being specifically expressed in skin andbreast tissue. [See EXAMPLES 54 and 55 for protocol]

(c) Adult Skin Cell Proliferation:

Procedure: Skin cells were isolated from 16 week old animals (2 wildtype and 4 homozygous mice). These were developed into primaryfibroblast cultures and the fibroblast proliferation rates were measuredin a strictly controlled protocol. The ability of this assay to detecthyper-proliferative and hypo-proliferative phenotypes has beendemonstrated with p53 and Ku80. Proliferation was measured using Brduincorporation.

Specifically, in these studies the skin fibroblast proliferation assaywas used. An increase in the number of cells in a standardized culturewas used as a measure of relative proliferative capacity. Primaryfibroblasts were established from skin biopsies taken from wild type andmutant mice. Duplicate or triplicate cultures of 0.05 million cells wereplated and allowed to grow for six days. At the end of the cultureperiod, the number of cells present in the culture was determined usinga electronic particle counter.

Results:

The female (−/−) mice exhibited a notably decreased mean skin fibroblastproliferation rate when compared with their gender-matched (+/+)littermates.

Thus, homozygous mutant mice demonstrated a hypo-proliferativephenotype. As suggested by these observations, antagonists or inhibitorsof PRO1550 polypeptides would mimic this hypo-proliferative phenotypeand could function as tumor suppressors and would be useful indecreasing abnormal cell proliferation. Thus, UNQ762 plays a role infibroblast activation and migration.

(d) Phenotypic Analysis: Metabolism—Blood Chemistry

In the area of metabolism, targets may be identified for the treatmentof diabetes. Blood chemistry phenotypic analysis includes blood glucosemeasurements. The COBAS Integra 400 (mfr: Roche) was used for runningblood chemistry tests on the mice. In addition to measuring bloodglucose levels the following blood chemistry tests are also routinelyperformed: Alkaline Phosphatase; Alanine Amino-Transferase; Albumin;Bilirubin; Phosphorous; Creatinine; BUN=Blood Urea Nitrogen; Calcium;Uric Acid; Sodium; Potassium; and Chloride. In the area of metabolism,targets may be identified for the treatment of diabetes. Blood chemistryphenotypic analysis includes glucose tolerance tests to measure insulinsensitivity and changes in glucose metabolism. Abnormal glucosetolerance test results may indicate but may not be limited to thefollowing disorders or conditions: Diabetes Type 1 and Type 2, SyndromeX, various cardiovascular diseases and/or obesity.

Results:

Both the male and female (−/−) mice exhibited notably increased meanserum alkaline phosphatase levels when compared with theirgender-matched (+/+) littermates and the historical means. There is alsoelevation of alkaline phosphatase in heterozygous (+/−) animals,particularly males.

48.24. Generation and Analysis of Mice Comprising DNA73730-1679 (UNQ777)Gene Disruptions

In these knockout experiments, the gene encoding PRO1571 polypeptides(designated as DNA73730-1679) (UNQ777) was disrupted. The gene specificinformation for these studies is as follows: the mutated mouse genecorresponds to nucleotide reference: NM_(—)019500 Mus musculus claudin14 (Cldn14); protein reference: Q9ZOS3 ACCESSION: Q9ZOS3 NID: Musmusculus (Mouse). Claudin-14; the human gene sequence reference:NM_(—)144492 ACCESSION: NM_(—)144492 NID: gi 21536293 ref NM_(—)144492.1Homo sapiens claudin 14 (CLDN14), transcript variant 1; the humanprotein sequence corresponds to reference: 095500 ACCESSION:095500 NID:Homo sapiens (Human). Claudin-14.

The mouse gene of interest is Cldn14 (claudin 14), ortholog of humanCLDN14. Aliases include DFNB29.

CLDN14 is an integral plasma membrane protein that likely functions asan adhesion molecule and component of tight junctions, structures thatform a physical barrier around epithelial or endothelial cells. CLDN14interacts with complementary proteins on adjacent cells and with itself,forming a lateral copolymer. Tight junctions prevent the movement ofwater and solutes through paracellular spaces as well as the movement ofplasma membrane proteins between the apical and basolateral or abluminalsurfaces of epithelial or endothelial cells. Tight junctions alsorecruit cytoskeletal proteins and signaling molecules and likelyparticipate in signal transduction processes (Gonzalez-Mariscal et al,Prog Biophys Mol Biol 81(1): 1-44 (2003); Tsukita et al, Nat Rev MolCell Biol 2(4):285-93 (2001); Heiskala et al, Traffic 2(2):93-8 (2001)).As a component of tight junctions, CLDN14 likely plays a role inparacellular transport and cellular asymmetry. Expression of CLDN14 isevident in cochlear inner and outer hair cells and supporting cells, inthe collecting ducts of the kidney, and around the lobules of the liver(Yosef et al, Hum Mol Genet 12(16):2049-61 (2003)). Mutations in CLDN14cause deafness in humans (Wilcox et al, Cell 104(1):165-72 (2001)).

Ben-Yosef and colleagues (2003) investigated the physiological role ofCLDN14 using knockout mice. They showed that the CLDN14 (−/−) micedisplayed rapid degeneration of cochlear outer hair cells, slowdegeneration of inner hair cells, and decreased paracellularpermeability for cations, resulting in deafness. They also showed thatCLDN14 is expressed in tight junctions of hair cells and supportingcells. Ben-Yosef and colleagues concluded that CLDN14 is required forrestricting paracellular transport of cations, which is important formaintaining the proper ionic composition of the fluid surrounding thebasolateral surface of outer hair cells.

Targeted or gene trap mutations are generated in strain129SvEv^(Brd)-derived embryonic stem (ES) cells. The chimeric mice arebred to C57BL/6J albino mice to generate F1 heterozygous animals. Theseprogeny are intercrossed to generate F2 wild type, heterozygous, andhomozygous mutant progeny. On rare occasions, for example when very fewF1 mice are obtained from the chimera, F1 heterozygous mice are crossedto 129SvEv^(Brd)/C57 hybrid mice to yield additional heterozygousanimals for the intercross to generate the F2 mice. Level I phenotypicanalysis is performed on mice from this generation

wt het hom Total Observed 16 40 26 82 Expected 20.5 41 20.5 82 Chi-Sq. =0.67 Significance = 0.71533805 (hom/n) = 0.27 Avg. Litter Size = 9Mutation InformationMutation Type: Homologous Recombination (standard)Description: Coding exon 1 was targeted (NCBI accession NM_(—)019500.3).1. Wild-type Expression Panel: Expression of the target gene wasdetected in brain, spinal cord, and eye among the 13 adult tissuesamples tested by RT-PCR.2. QC Expression: QC Images: Disruption of the target gene was confirmedby Southern hybridization analysis.

48.24.1. Phenotypic Analysis (for Disrupted Gene: DNA73730-1679 (UNQ777)

(a) Overall Phenotypic Summary:

Mutation of the gene encoding the ortholog of human claudin 14 (CLDN14)resulted in hearing impaired (−/−) mice, exhibiting cochlear hair celldegeneration. Microscopic analysis revealed degeneration and loss ofsensory cochlear hair cells in the inner ear of the homozygous mutantmice, confirming the hearing impairment noted during prepulse inhibitiontesting. In addition, the mutants exhibited an increased mean plateletcount and an increased subsets of CD4 and CD8 cells when compared withthat of their wild-type littermates and the historical mean. The mutant(−/−) mice also exhibited decreased bone related measurements withdecreased vertebral bone volume, number and connectivity density. Themutant (−/−) mice exhibited increased mean serum cholesterol andtriglyceride levels. Disruption of the target gene was confirmed bySouthern hybridization analysis.

(b) Expression:

Claudin 14 is a tight junction protein implicated in hearing functionfor its expression in the cochlea. Claudin 14's increased expression isalso associated with synovial macrophages in rheumatoid arthritis andtherefore plays an important role in the immune system.

(c) Pathology

Microscopic: The (−/−) mice exhibited diffuse marked degenerationsensory cochlear hair cells in the inner ear, characterized by acomplete loss of both inner and outer cochlear hair cells on the basilarmembrane.

Gene Expression: LacZ activity was not detected in the panel of tissuesby immunohistochemical analysis.

(d) Immunology Phenotypic Analysis

Immune related and inflammatory diseases are the manifestation orconsequence of fairly complex, often multiple interconnected biologicalpathways which in normal physiology are critical to respond to insult orinjury, initiate repair from insult or injury, and mount innate andacquired defense against foreign organisms. Disease or pathology occurswhen these normal physiological pathways cause additional insult orinjury either as directly related to the intensity of the response, as aconsequence of abnormal regulation or excessive stimulation, as areaction to self, or as a combination of these.

Though the genesis of these diseases often involves multistep pathwaysand often multiple different biological systems/pathways, interventionat critical points in one or more of these pathways can have anameliorative or therapeutic effect. Therapeutic intervention can occurby either antagonism of a detrimental process/pathway or stimulation ofa beneficial process/pathway.

T lymphocytes (T cells) are an important component of a mammalian immuneresponse. T cells recognize antigens which are associated with aself-molecule encoded by genes within the major histocompatibilitycomplex (MHC). The antigen may be displayed together with MHC moleculeson the surface of antigen presenting cells, virus infected cells, cancercells, grafts, etc. The T cell system eliminates these altered cellswhich pose a health threat to the host mammal. T cells include helper Tcells and cytotoxic T cells. Helper T cells proliferate extensivelyfollowing recognition of an antigen-MHC complex on an antigen presentingcell. Helper T cells also secrete a variety of cytokines, i.e.,lymphokines, which play a central role in the activation of B cells,cytotoxic T cells and a variety of other cells which participate in theimmune response.

In many immune responses, inflammatory cells infiltrate the site ofinjury or infection. The migrating cells may be neutrophilic,eosinophilic, monocytic or lymphocytic as can be determined byhistological examination of the affected tissues. Current Protocols inImmunology, ed. John E. Coligan, 1994, John Wiley & Sons, Inc.

Many immune related diseases are known and have been extensivelystudied. Such diseases include immune-mediated inflammatory diseases(such as rheumatoid arthritis, immune mediated renal disease,hepatobiliary diseases, inflammatory bowel disease (IBD), psoriasis, andasthma), non-immune-mediated inflammatory diseases, infectious diseases,immunodeficiency diseases, neoplasia, and graft rejection, etc. In thearea of immunology, targets were identified for the treatment ofinflammation and inflammatory disorders.

In the area of immunology, targets have been identified herein for thetreatment of inflammation and inflammatory disorders. Immune relateddiseases, in one instance, could be treated by suppressing the immuneresponse. Using neutralizing antibodies that inhibit molecules havingimmune stimulatory activity would be beneficial in the treatment ofimmune-mediated and inflammatory diseases. Molecules which inhibit theimmune response can be utilized (proteins directly or via the use ofantibody agonists) to inhibit the immune response and thus ameliorateimmune related disease.

The following tests were performed:

Hematology Analysis:

Test Description: Blood tests are carried out by Abbott's Cell-Dyn3500R, an automated hematology analyzer. Some of its features include afive-part WBC differential. ‘Patient’ reports can cover over 22parameters in all.

Results:

Hematology: The (+/−) mice exhibited an increased mean platelet countwhen compared with that of their (+/+) littermates and the historicalmean.

Thus, mutant mice deficient in the DNA73730-1679 gene resulted in aphenotype related to coagulation disorders. In this regard, inhibitorsor antagonists of PRO1571 polypeptides would be useful in treatingdisorders related to abnormal blood coagulation such as hemophilia.

Fluorescence-Activated Cell-Sorting (FACS) Analysis

Procedure:

FACS analysis of immune cell composition from peripheral blood wasperformed including CD4, CD8 and T cell receptor to evaluate Tlymphocytes, CD19 for B lymphocytes, CD45 as a leukocyte marker and panNK for natural killer cells. The FACS analysis was carried out on 2 wildtype and 6 homozygous mice and included cells derived from thymus,spleen, bone marrow and lymph node.

In these studies, analyzed cells were isolated from thymus, peripheralblood, spleen, bone marrow and lymph nodes. Flow cytometry was designedto determine the relative proportions of CD4 and CD8 positive T cells, Bcells, NK cells and monocytes in the mononuclear cell population. ABecton-Dickinson FACS Calibur 3-laser FACS machine was used to assessimmune status. For Phenotypic Assays and Screening, this machine recordsCD4+/CD8−, CD8+/CD4−, NK, B cell and monocyte numbers in addition to theCD4+/CD8+ ratio.

The mononuclear cell profile was derived by staining a single sample oflysed peripheral blood from each mouse with a panel of sixlineage-specific antibodies: CD45 PerCP, anti-TCRb APC, CD4 PE, CD8FITC, pan-NK PE, and CD19 FITC. The two FITC and PE labeled antibodiesstain mutually exclusive cell types. The samples were analyzed using aBecton Dickinson FACS Calibur flow cytometer with CellQuest software.

Results:

The (−/−) mice exhibited an altered distribution of leukocyte subsets inthe peripheral blood, characterized by an increased mean percentages ofCD62hi, CD44int (subsets of CD4 and CD8) cells in the cell populationwhen compared with their (+/+) littermates and the historical means.

Thus, knocking out the gene which encodes PRO1571 polypeptides causes anincrease in the T cell population. From these observations, PRO1571polypeptides or the gene encoding PRO1571 appears to act as a negativeregulator of T cell proliferation. Thus, antagonists or inhibitors ofPRO1571 polypeptides would be beneficial in enhancing T cellproliferation.

(e) Bone Metabolism & Body Diagnostics: Radiology Phenotypic Analysis

In the area of bone metabolism, targets were identified herein for thetreatment of arthritis, osteoporosis, osteopenia and osteopetrosis aswell as identifying targets that promote bone healing. Tests included:

DEXA for measurement of bone mineral density on femur and vertebra

MicroCT for very high resolution and very high sensitivity measurementsof bone mineral density for both trabecular and cortical bone.

Dexa Analysis—Test Description:

Procedure: A cohort of 4 wild type, 4 heterozygous and 8 homozygous micewere tested in this assay. Dual Energy X-ray Absorptiometry (DEXA) hasbeen used successfully to identify changes in bone. Anesthetized animalswere examined and bone mineral content (BMC), BMC/LBM ratios, volumetricbone mineral density (vBMD), total body BMD, femur BMD and vertebra BMDwere measured.

The mouse was anesthetized by intraperitoneal injection of Avertin(1.25% 2,2,2,-tribromoethanol, 20 ml/kg body weight), body length andweight were measured, and then the mouse was placed in a prone positionon the platform of the PIXImus™ Densitometer (Lunar Inc.) for a DEXAscan. Using Lunar PIXImus software, the bone mineral density (BMD) andfat composition (% fat) and total tissue mass (TTM) were determined inthe regions of interest (ROI) [i.e., whole body, vertebrae, and bothfemurs].

Bone MicroCT Analysis:

Procedure: MicroCT was also used to get very sensitive measurements ofBMD. One vertebra and 1 femur were taken from a cohort of 4 wild typeand 8 homozygous mice. Measurements were taken of lumbar 5 vertebratrabecular bone volume, trabecular thickness, connectivity density andmidshaft femur total bone area and cortical thickness. The μCT40 scansprovided detailed information on bone mass and architecture. Multiplebones were placed into sample holders and scanned automatically.Instrument software was used to select regions of interest for analysis.Trabecular bone parameters were analyzed in the fifth lumbar vertebrae(LV5) at 16 micrometer resolution and cortical bone parameters wereanalyzed in the femur midshaft at a resolution of 20 micrometers.

Results:

Micro CT: The male (−/−) mice exhibited decreased mean vertebraltrabecular bone volume, number, and connectivity density when comparedwith that of their gender-matched (+/+) littermates and the historicalmeans.

The (−/−) mice analyzed by Micro CT analysis exhibited decreased bonemeasurements when compared with their (+/+) littermates, suggestive ofabnormal bone disorders. The negative bone phenotype indicates thatPRO1571 polypeptides or agonists thereof would be useful for maintainingbone homeostasis. In addition, PRO1571 polypeptides would be useful inbone healing or for the treatment of arthritis or osteoporosis, whereasantagonists (or inhibitors) of PRO1571 polypeptides or its encoding genewould lead to abnormal or pathological bone disorders includinginflammatory diseases associated with abnormal bone metabolism includingarthritis, osteoporosis and osteopenia.

(f) Phenotypic Analysis: CNS/Neurology

In the area of neurology, analysis focused herein on identifying in vivovalidated targets for the treatment of neurological and psychiatricdisorders including depression, generalized anxiety disorders, attentiondeficit hyperactivity disorder, obsessive compulsive disorder,schizophrenia, cognitive disorders, hyperalgesia and sensory disorders.Neurological disorders include the category defined as “anxietydisorders” which include but are not limited to: mild to moderateanxiety, anxiety disorder due to a general medical condition, anxietydisorder not otherwise specified, generalized anxiety disorder, panicattack, panic disorder with agoraphobia, panic disorder withoutagoraphobia, posttraumatic stress disorder, social phobia, specificphobia, substance-induced anxiety disorder, acute alcohol withdrawal,obsessive compulsive disorder, agoraphobia, bipolar disorder I or II,bipolar disorder not otherwise specified, cyclothymic disorder,depressive disorder, major depressive disorder, mood disorder,substance-induced mood disorder. In addition, anxiety disorders mayapply to personality disorders including but not limited to thefollowing types: paranoid, antisocial, avoidant behavior, borderlinepersonality disorders, dependent, histronic, narcissistic,obsessive-compulsive, schizoid, and schizotypal.

Procedure:

Behavioral screens were performed on a cohort of 4 wild type, 4heterozygous and 8 homozygous mutant mice. All behavioral tests weredone between 12 and 16 weeks of age unless reduced viabilitynecessitates earlier testing. These tests included open field to measureanxiety, activity levels and exploration.

Prepulse Inhibition of the Acoustic Startle Reflex

Prepulse inhibition of the acoustic startle reflex occurs when a loud120 decibel (dB) startle-inducing tone is preceded by a softer(prepulse) tone. The PPI paradigm consists of six different trial types(70 dB background noise, 120 dB alone, 74 dB+120 dB−pp4, 78 dB+120dB−pp8, 82 dB+120 dB−pp12, and 90 dB+120 dB−pp20) each repeated inpseudo random order six times for a total of 36 trials. The max responseto the stimulus (V max) is averaged for each trial type. Animals with a120 dB average value equal to or below 100 are excluded from analysis.The percent that the prepulse inhibits the animal's response to thestartle stimulus is calculated and graphed.

Results:

PPI: All 8 (−/−) mice failed to exhibit a startle response, suggestinghearing impairment in the mutants. Therefore, prepulse inhibition couldnot be assessed. These results are consistent with observation that the(−/−) mice exhibited diffuse marked degeneration sensory cochlear haircells in the inner ear, characterized by a complete loss of both innerand outer cochlear hair cells on the basilar membrane.

(g) Phenotypic Analysis: Cardiology

In the area of cardiovascular biology, targets were identified hereinfor the treatment of hypertension, atherosclerosis, heart failure,stroke, various coronary artery diseases, dyslipidemias such as highcholesterol (hypercholesterolemia) and elevated serum triglycerides(hypertriglyceridemia), diabetes and/or obesity. The phenotypic testsincluded the measurement of serum cholesterol and triglycerides.

Blood Lipids

Procedure: A cohort of 4 wild type, 4 heterozygous and 8 homozygous micewere tested in this assay. High cholesterol levels and increasedtriglyceride blood levels are recognized risk factors in the developmentof cardiovascular disease and/or diabetes. Measuring blood lipidsfacilitates the finding of biological switches that regulate blood lipidlevels. Inhibition of factors which elevate blood lipid levels may beuseful for reducing the risk for cardiovascular disease. In these bloodchemistry tests, measurements were recorded using the COBAS Integra 400(mfr: Roche).

Results:

Blood Chemistry: The male (−/−) mice exhibited increased mean serumcholesterol and triglyceride levels when compared with that of theirgender-matched (+/+) littermates and the historical mean.

As summarized above, the (−/−) mice exhibited increased mean serumcholesterol and triglyceride levels when compared with theirgender-matched (+/+) littermates and the historical means. Thus, mutantmice deficient in the PRO1571 gene may serve as a model forcardiovascular disease. PRO1571 polypeptides or its encoding gene wouldbe useful in regulating blood lipids such as cholesterol andtriglycerides. Thus, PRO1571 polypeptides or agonists thereof would beuseful in the treatment of such cardiovascular diseases as hypertension,atherosclerosis, heart failure, stroke, various coronary diseases,hypercholesterolemia, hypertriglyceridemia, diabetes and/or obesity.

48.25. Generation and Analysis of Mice Comprising DNA73734-1680 (UNQ778)Gene Disruptions

In these knockout experiments, the gene encoding PRO1572 polypeptides(designated as DNA73734-1680) (UNQ778) was disrupted. The gene specificinformation for these studies is as follows: the mutated mouse genecorresponds to nucleotide reference: NM_(—)019815 ACCESSION:NM_(—)019815 NID: gi 9790074 ref NM_(—)019815.1 Mus musculus claudin 18(Cldn18); protein reference: P56857 ACCESSION:P56857 NID: Mus musculus(Mouse). Claudin-18; the human gene sequence reference: NM_(—)016369Homo sapiens claudin 18 (CLDN18), transcript variant 1; the humanprotein sequence corresponds to reference: P56856 ACCESSION:P56856 NID:Homo sapiens (Human). Claudin-18.

The mouse gene of interest is Cldn18 (claudin 18), ortholog of humanCLDN18.

CLDN18 is an integral plasma membrane protein expressed primarily inlung and stomach epithelial cells that functions as a component of tightjunctions. CLDN18 likely plays a role in paracellular transport and cellpolarity (Niimi et al, Mol Cell Biol 21(21):7380-90 (2001); Heiskala etal, Traffic 2(2):93-8 (2001); Gonzalez-Mariscal et al, Prog Biophys MolBiol 81(1):1-44 (2003)).

Targeted or gene trap mutations are generated in strain129SvEv^(Brd)-derived embryonic stem (ES) cells. The chimeric mice arebred to C57BL/6J albino mice to generate F1 heterozygous animals. Theseprogeny are intercrossed to generate F2 wild type, heterozygous, andhomozygous mutant progeny. On rare occasions, for example when very fewF1 mice are obtained from the chimera, F1 heterozygous mice are crossedto 129SvEv^(Brd)/C57 hybrid mice to yield additional heterozygousanimals for the intercross to generate the F2 mice. Level I phenotypicanalysis is performed on mice from this generation

wt het hom Total Observed 24 37 23 84 Expected 21 42 21 84 Chi-Sq. =0.33 Significance = 0.8478937 (hom/n) = 0.24 Avg. Litter Size = 9Mutation InformationMutation Type: Homologous Recombination (standard)Description: Coding exons 2 through 4 were targeted (NCBI accessionNM_(—)019815.1).1. Wild-type Expression Panel: Expression of the target gene wasdetected in spinal cord; lung; kidney; stomach, small intestine, andcolon; and asthmatic lung among 26 adult tissue samples tested byRT-PCR.2. QC Expression: QC Images: Disruption of the target gene was confirmedby Southern hybridization analysis.

48.25.1. Phenotypic Analysis (for Disrupted Gene: DNA73734-1680 (UNQ778)

(a) Overall Phenotypic Summary:

Mutation of the gene encoding the ortholog of human claudin 18 (CLDN18)resulted in decreased bone mineral content and density measurements in(−/−) mice. Both the male and female homozygous mutant mice exhibitednotably decreased bone mineral content and density measurements whencompared with those of their gender-matched wild-type littermates andthe historical means. In addition, the homozygous mutants exhibitednumerous immunological abnormalities, including increased white bloodcell counts and an increased IL-6 response to LPS challenge. Necropsyrevealed thickened gastric mucosa with abnormal differentiation of thegastric gland epithelium and chronic inflammation in the homozygousmutant mice. Disruption of the target gene was confirmed by Southernhybridization analysis.

(b) Expression:

Claudin 18 has a unique expression pattern limited largely to the lungand stomach with relatively low expression in other tissues. Expressionis associated with rheumatoid arthritis with increased expression insynovial fibroblasts, macrophages and T cells.

(c) Pathology

Gross: The (−/−) mice exhibited markedly thickened gastric mucosa.

Microscopic: The (−/−) mice exhibited changes in the gastric mucosa,characterized by a marked loss of normal differentiation of gastricgland epithelium with decreased numbers of gastric chief cells andparietal cells and increased numbers of mucoid cells. Abnormal glandswith multiple branches were present, and moderate inflammatoryinfiltrates were also present in the mucosa and lamina propria andextended to the tops of the glandular mucosa. These observations areconsistent with specific expression in the stomach. GeneLogic data showsdecreased expression of UNQ778 in human gastric adenocarcinoma. In manyareas, the gastric glands contained numerous mucoid cells that replacedthe parietal and chief cells. There was also a marked reduction ineosinophilic cytoplasmic granules in the striated ducts of themandibular salivary glands in 2/3 male (−/−) mice.The (−/−) mice also exhibited an increase in stomach weight compared tothe (+/+) littermates.Gene Expression: LacZ activity was not detected in the panel of tissuesby immunohistochemical analysis.

(d) Immunology Phenotypic Analysis

Immune related and inflammatory diseases are the manifestation orconsequence of fairly complex, often multiple interconnected biologicalpathways which in normal physiology are critical to respond to insult orinjury, initiate repair from insult or injury, and mount innate andacquired defense against foreign organisms. Disease or pathology occurswhen these normal physiological pathways cause additional insult orinjury either as directly related to the intensity of the response, as aconsequence of abnormal regulation or excessive stimulation, as areaction to self, or as a combination of these.

Though the genesis of these diseases often involves multistep pathwaysand often multiple different biological systems/pathways, interventionat critical points in one or more of these pathways can have anameliorative or therapeutic effect. Therapeutic intervention can occurby either antagonism of a detrimental process/pathway or stimulation ofa beneficial process/pathway.

T lymphocytes (T cells) are an important component of a mammalian immuneresponse. T cells recognize antigens which are associated with aself-molecule encoded by genes within the major histocompatibilitycomplex (MHC). The antigen may be displayed together with MHC moleculeson the surface of antigen presenting cells, virus infected cells, cancercells, grafts, etc. The T cell system eliminates these altered cellswhich pose a health threat to the host mammal. T cells include helper Tcells and cytotoxic T cells. Helper T cells proliferate extensivelyfollowing recognition of an antigen-MHC complex on an antigen presentingcell. Helper T cells also secrete a variety of cytokines, i.e.,lymphokines, which play a central role in the activation of B cells,cytotoxic T cells and a variety of other cells which participate in theimmune response.

In many immune responses, inflammatory cells infiltrate the site ofinjury or infection. The migrating cells may be neutrophilic,eosinophilic, monocytic or lymphocytic as can be determined byhistological examination of the affected tissues. Current Protocols inImmunology, ed. John E. Coligan, 1994, John Wiley & Sons, Inc.

Many immune related diseases are known and have been extensivelystudied. Such diseases include immune-mediated inflammatory diseases(such as rheumatoid arthritis, immune mediated renal disease,hepatobiliary diseases, inflammatory bowel disease (IBD), psoriasis, andasthma), non-immune-mediated inflammatory diseases, infectious diseases,immunodeficiency diseases, neoplasia, and graft rejection, etc. In thearea of immunology, targets were identified for the treatment ofinflammation and inflammatory disorders.

In the area of immunology, targets have been identified herein for thetreatment of inflammation and inflammatory disorders. Immune relateddiseases, in one instance, could be treated by suppressing the immuneresponse. Using neutralizing antibodies that inhibit molecules havingimmune stimulatory activity would be beneficial in the treatment ofimmune-mediated and inflammatory diseases. Molecules which inhibit theimmune response can be utilized (proteins directly or via the use ofantibody agonists) to inhibit the immune response and thus ameliorateimmune related disease.

The following tests were performed:

Hematology Analysis:

Test Description: Blood tests are carried out by Abbott's Cell-Dyn3500R, an automated hematology analyzer. Some of its features include afive-part WBC differential. ‘Patient’ reports can cover over 22parameters in all.

Results:

Hematology: The (−/−) mice exhibited increased mean white blood cell,absolute neutrophil, absolute lymphocyte, and platelet counts whencompared with those of their (+/+) littermates and the historical means.

These results indicate that mutant (−/−) mice have several immunologicalabnormalities compared with their wild-type littermates. In summary, thehematology results indicate that the homozygous mutant mice exhibited anincreased white blood cell count, neutrophils and lymphocytes countcompared to their littermate controls indicating elevated levels ofprecursors of macrophages with increased phagocytic activity or abilityto engulf or kill extracellular pathogens. Thus, PRO1572 polypeptidesmust be essential for maintaining a normal immunological profileespecially for adaptive immunity. In addition, the mutant (−/−) miceexhibited an increased platelet count. Thus, mutant mice deficient inthe DNA73734-1680 gene resulted in a phenotype related to coagulationdisorders. In this regard, inhibitors or antagonists of PRO1572polypeptides would be useful in treating disorders related to abnormalblood coagulation such as hemophilia.

Fluorescence-Activated Cell-Sorting (FACS) Analysis

Procedure:

FACS analysis of immune cell composition from peripheral blood wasperformed including CD4, CD8 and T cell receptor to evaluate Tlymphocytes, CD19 for B lymphocytes, CD45 as a leukocyte marker and panNK for natural killer cells. The FACS analysis was carried out on 2 wildtype and 6 homozygous mice and included cells derived from thymus,spleen, bone marrow and lymph node.

In these studies, analyzed cells were isolated from thymus, peripheralblood, spleen, bone marrow and lymph nodes. Flow cytometry was designedto determine the relative proportions of CD4 and CD8 positive T cells, Bcells, NK cells and monocytes in the mononuclear cell population. ABecton-Dickinson FACS Calibur 3-laser FACS machine was used to assessimmune status. For Phenotypic Assays and Screening, this machine recordsCD4+/CD8−, CD8+/CD4−, NK, B cell and monocyte numbers in addition to theCD4+/CD8+ ratio.

The mononuclear cell profile was derived by staining a single sample oflysed peripheral blood from each mouse with a panel of sixlineage-specific antibodies: CD45 PerCP, anti-TCRb APC, CD4 PE, CD8FITC, pan-NK PE, and CD19 FITC. The two FITC and PE labeled antibodiesstain mutually exclusive cell types. The samples were analyzed using aBecton Dickinson FACS Calibur flow cytometer with CellQuest software.

Results:

A significant decrease in CD117 cells were observed in the peritoneallavage in the mutant (−/−) mice compared to the wild-type (+/+)littermates. Thus, hematopoietic progenitors are decreased in theseknockout mice. Thus, the gene encoding PRO1572 polypeptides must beessential for hematopoietic progenitor development and/or production.

Acute Phase Response:

Test Description: Bacterial lipopolysaccharide (LPS) is an endotoxin,and as such is a potent inducer of an acute phase response and systemicinflammation. The Level I LPS mice were injected intraperitoneally(i.p.) with a sublethal dose of LPS in 200 μL sterile saline using a 26gauge needle. The doses were based on the average weight of the micetested at 1 μg/g body weight 3 hours after injection; a 100 ul bloodsample was then taken and analyzed for the presence of TNFa, MCP-1, andIL-6 on the FACS Calibur instrument.

Results:

Acute Phase Response: The (−/−) mice exhibited an increased IL-6response to LPS challenge when compared with that of their (+/+)littermates and the historical mean.

In summary, the LPS endotoxin challenge demonstrated that knockout micedeficient in the gene encoding PRO1572 polypeptides exhibitimmunological abnormalities when compared with their wild-typelittermates. In particular, the mutant mice exhibited an increasedability to elicit an immunological response (IL-6 production) whenchallenged with the LPS endotoxin indicating a proinflammatory response.IL-6 contributes to the later stages of B cell activation. In addition,IL-6 plays a critical role in inducing the acute phase response andsystemic inflammation. This suggests that inhibitors or antagonists toPRO1572 polypeptides would stimulate the immune system and would findutility in the cases wherein this effect would be beneficial to theindividual such as in the case of leukemia, and other types of cancer,and in immuno-compromised patients, such as AIDS sufferers. Accordingly,PRO1572 polypeptides or agonists thereof would be useful in inhibitingthe immune response and would be useful candidates for suppressingharmful immune responses, e.g. in the case of graft rejection orgraft-versus-host diseases.

(e) Bone Metabolism & Body Diagnostics: Radiology Phenotypic Analysis

In the area of bone metabolism, targets were identified herein for thetreatment of arthritis, osteoporosis, osteopenia and osteopetrosis aswell as identifying targets that promote bone healing. Tests included:

DEXA for measurement of bone mineral density on femur and vertebra

MicroCT for very high resolution and very high sensitivity measurementsof bone mineral density for both trabecular and cortical bone.

Dexa Analysis—Test Description:

Procedure: A cohort of 4 wild type, 4 heterozygous and 8 homozygous micewere tested in this assay. Dual Energy X-ray Absorptiometry (DEXA) hasbeen used successfully to identify changes in bone. Anesthetized animalswere examined and bone mineral content (BMC), BMC/LBM ratios, volumetricbone mineral density (vBMD), total body BMD, femur BMD and vertebra BMDwere measured.

The mouse was anesthetized by intraperitoneal injection of Avertin(1.25% 2,2,2,-tribromoethanol, 20 ml/kg body weight), body length andweight were measured, and then the mouse was placed in a prone positionon the platform of the PIXImus™ Densitometer (Lunar Inc.) for a DEXAscan. Using Lunar PIXImus software, the bone mineral density (BMD) andfat composition (% fat) and total tissue mass (TTM) were determined inthe regions of interest (ROI) [i.e., whole body, vertebrae, and bothfemurs].

Bone MicroCT Analysis:

Procedure: MicroCT was also used to get very sensitive measurements ofBMD. One vertebra and 1 femur were taken from a cohort of 4 wild typeand 8 homozygous mice. Measurements were taken of lumbar 5 vertebratrabecular bone volume, trabecular thickness, connectivity density andmidshaft femur total bone area and cortical thickness. The μCT40 scansprovided detailed information on bone mass and architecture. Multiplebones were placed into sample holders and scanned automatically.Instrument software was used to select regions of interest for analysis.Trabecular bone parameters were analyzed in the fifth lumbar vertebrae(LV5) at 16 micrometer resolution and cortical bone parameters wereanalyzed in the femur midshaft at a resolution of 20 micrometers.

Results:

DEXA: The (−/−) mice exhibited notably decreased mean bone mineralcontent and density measurements when compared with those of theirgender-matched (+/+) littermates and the historical means.

Micro CT: The male (−/−) mice exhibited notably decreased mean femoralmid-shaft cortical thickness, trabecular bone volume and trabecular bonethickness when compared with that of their gender-matched (+/+)littermates and the historical mean.

The (−/−) mice analyzed by DEXA and bone micro CT analysis exhibiteddecreased bone measurements when compared with their (+/+) littermates,suggestive of abnormal bone disorders. The (−/−) mice exhibited anegative bone phenotype with abnormal and decreased bone measurementsreflective of bone metabolic disorders. The negative bone phenotypeindicates that PRO1572 polypeptides or agonists thereof would be usefulfor maintaining bone homeostasis. In addition, PRO1572 polypeptideswould be useful in bone healing or for the treatment of arthritis orosteoporosis, whereas antagonists (or inhibitors) of PRO1572polypeptides or its encoding gene would lead to abnormal or pathologicalbone disorders including inflammatory diseases associated with abnormalbone metabolism including arthritis, osteoporosis and osteopenia.

48.26. Generation and Analysis of Mice Comprising DNA76531-1701 (UNQ832)Gene Disruptions

In these knockout experiments, the gene encoding PRO1759 polypeptides(designated as DNA76531-1701) (UNQ832) was disrupted. The gene specificinformation for these studies is as follows: the mutated mouse genecorresponds to nucleotide reference: NM_(—)134100 Mus musculus DNASegment, Chr 15, Mouse Genome Informatics 27 (D15Mgi27); proteinreference: Q921Y4 ACCESSION: Q921Y4 NID: Mus musculus (Mouse). D15Mgi27protein (Mus musculus NOD-derived CD11c ve dendritic cells cDNA, RIKENfull-length enriched library, clone:F630109H06 product:hypotheticalGeneral substrate transporters containing protein, full insertsequence); the human gene sequence reference: NM_(—)032889 Homo sapienshypothetical protein MGC11308 (MGC11308); the human protein sequencecorresponds to reference: Q961A5 ACCESSION: Q961A5 NID: Homo sapiens(Human). UNKNOWN (PROTEIN FOR MGC:111308).

The mouse gene of interest is D15Mgi27 (DNA Segment, Chr 15, MouseGenome Informatics 27), ortholog of human hypothetical protein MGC11308.

Hypothetical protein MGC11308 is a putative integral plasma membraneprotein (Clark et al, Genome Res 13(10):2265-70 (2003)) and “majorfacilitator superfamily” (MFS) member that likely functions as atransporter (Pao et al, Microbiol Mol Biol Rev 62(1):1-34 (1998)). Theprotein consists primarily of a signal peptide and 10 transmembranesegments within a DUF791 (“protein of unknown function”) domain (Pfamaccession PF05631).

Targeted or gene trap mutations are generated in strain129SvEv^(Brd)-derived embryonic stem (ES) cells. The chimeric mice arebred to C57BL/6J albino mice to generate F1 heterozygous animals. Theseprogeny are intercrossed to generate F2 wild type, heterozygous, andhomozygous mutant progeny. On rare occasions, for example when very fewF1 mice are obtained from the chimera, F1 heterozygous mice are crossedto 129SvEv^(Brd)/C57 hybrid mice to yield additional heterozygousanimals for the intercross to generate the F2 mice. Level I phenotypicanalysis is performed on mice from this generation

wt het hom Total Observed 18 32 18 68 Expected 17 34 17 68 Chi-Sq. =1.14 Significance = 0.5655255 (hom/n) = 0.26 Avg. Litter Size = 9Mutation InformationMutation Type Homologous Recombination (standard)Description: Coding exon 1 was targeted (NCBI accession NM_(—)134100.2).1. Wild-type Expression Panel: Expression of the target gene wasdetected in embryonic stem (ES) cells and in all 26 adult tissue samplestested by RT-PCR, except bone.2. QC Expression: QC Images: Disruption of the target gene was confirmedby Southern hybridization analysis.

48.26.1. Phenotypic Analysis (for Disrupted Gene: DNA76531-1701 (UNQ832)

(a) Overall Phenotypic Summary:

Mutation of the gene encoding the ortholog of human hypothetical proteinMGC11308 resulted in a decreased depressive-like response in (−/−) mice.Gene disruption was confirmed by Southern blot.

(b) Phenotypic Analysis: CNS/Neurology

In the area of neurology, analysis focused herein on identifying in vivovalidated targets for the treatment of neurological and psychiatricdisorders including depression, generalized anxiety disorders, attentiondeficit hyperactivity disorder, obsessive compulsive disorder,schizophrenia, cognitive disorders, hyperalgesia and sensory disorders.Neurological disorders include the category defined as “anxietydisorders” which include but are not limited to: mild to moderateanxiety, anxiety disorder due to a general medical condition, anxietydisorder not otherwise specified, generalized anxiety disorder, panicattack, panic disorder with agoraphobia, panic disorder withoutagoraphobia, posttraumatic stress disorder, social phobia, specificphobia, substance-induced anxiety disorder, acute alcohol withdrawal,obsessive compulsive disorder, agoraphobia, bipolar disorder I or II,bipolar disorder not otherwise specified, cyclothymic disorder,depressive disorder, major depressive disorder, mood disorder,substance-induced mood disorder. In addition, anxiety disorders mayapply to personality disorders including but not limited to thefollowing types: paranoid, antisocial, avoidant behavior, borderlinepersonality disorders, dependent, histronic, narcissistic,obsessive-compulsive, schizoid, and schizotypal.

Procedure:

Behavioral screens were performed on a cohort of 4 wild type, 4heterozygous and 8 homozygous mutant mice. All behavioral tests weredone between 12 and 16 weeks of age unless reduced viabilitynecessitates earlier testing. These tests included open field to measureanxiety, activity levels and exploration.

Functional Observational Battery (FOB) Test—Tail Suspension Testing:

The FOB is a series of situations applied to the animal to determinegross sensory and motor deficits. A subset of tests from the Irwinneurological screen that evaluates gross neurological function is used.In general, short-duration, tactile, olfactory, and visual stimuli areapplied to the animal to determine their ability to detect and respondnormally. These simple tests take approximately 10 minutes and the mouseis returned to its home cage at the end of testing.

Tail Suspension Testing:

The tail suspension test is a procedure that has been developed as amodel for depressive-like behavior in rodents. In this particular setup,a mouse is suspended by its tail for 6 minutes, and in response themouse will struggle to escape from this position. After a certain periodof time the struggling of the mouse decreases and this is interpreted asa type of learned helplessness paradigm. Animals with invalid data (i.e.climbed their tail during the testing period) are excluded fromanalysis.

Results:

Tail Suspension2: The (−/−) mice exhibited a decreased median immobilitytime when compared with that of their (+/+) littermates and thehistorical mean, suggesting a decreased depressive-like response in themutants.

In summary, the tail suspension testing revealed a phenotype associatedwith increased anxiety which could be associated with mild to moderateanxiety, anxiety due to a general medical condition, and/or bipolardisorders; hyperactivity; sensory disorders; obsessive-compulsivedisorders, schizophrenia or a paranoid personality. Thus, PRO1759polypeptides or agonists thereof would be useful in the treatment ofsuch neurological disorders.

48.27. Generation and Analysis of Mice Comprising DNA82372 (UNQ886) GeneDisruptions

In these knockout experiments, the gene encoding PRO1904 polypeptides(designated as DNA82372) (UNQ886) was disrupted. The gene specificinformation for these studies is as follows: the mutated mouse genecorresponds to nucleotide reference: MIS UNQ886 LGID:15208; proteinreference: MIS_UNQ886 ORF (LGID:15208); the human gene sequencereference: NM_(—)004590 Homo sapiens chemokine (C-C motif) ligand 16(CCL16); the human protein sequence corresponds to reference: 015467ACCESSION:015467 NID: Homo sapiens (Human). Small inducible cytokine A16precursor (CCL16) (IL-10-inducible chemokine) (Chemokine LEC)(Liver-expressed chemokine) (Monotactin-1) (MTN-1) (Chemokine CC-4)(HCC-4) (NCC-4) (Lymphocyte and monocyte chemoattractant) (LMC) (LCC-1).

The mouse gene of interest is represented by a predicted transcript(Lexicon accession: MIS_UNQ886), which is orthologous with human CCL16(chemokine [C-C motif] ligand 16). Aliases include LEC, LMC, NCC4,CKb12, HCC-4, LCC-1, Mtn-1, NCC-4, SCYL4, ILINCK, SCYA16, monotactin-1,chemokine LEC, chemokine CC-4, new CC chemokine 4, IL-10-induciblechemokine, liver-expressed chemokine, liver CC chemokine-1 precursor,and lymphocyte and monocyte chemoattractant.

CCL16 is a secreted protein that functions as a low-affinity ligand forcytokine receptors CCR1, CCR2, CCR5, and CCR8 (Howard et al, Blood96(3):840-5 (2000); Nomiyama et al, Int Immunol 13(8): 1021-9 (2001))and as a high-affinity ligand for histamine receptor H4 expressed oneosinophils (Nakayama et al, J Immunol 173(3):2078-83 (2004)). CCL16 isexpressed constitutively by hepatocytes (Nomiyama et al, Int Immunol13(8):1021-9 (2001); Shoudai et al, Biochim Biophys Acta 1396(3):273-7(1998)), and CCL16 expression is upregulated by interleukin-10 inactivated monocytes (Hedrick et al, Blood 91(11):4242-7 (1998)). CCL16is a regulator of immune cell function. CCL16 stimulates chemotaxis ofeosinophils, monocytes, T-cells, and dendritic cells, enhances thefunction of macrophages, and augments the lytic activity of T-cells(Nakayama et al, J Immunol 173(3):2078-83 (2004); Guiducci et al, JImmunol 172(7):4026-36 (2004); Cappello et al, J Leukoc Biol 75(1):135-42 (2004)). CCL16 may play a role in angiogenesis by triggeringangiogenic activities in endothelial cells (Strasly et al, Blood103(1):40-9 (2004)). CCL16 can delay tumor growth and inhibitmetastasis, suggesting that the cytokine may be useful for treatment ofcertain types of cancer (Li et al, Cancer Res 63(23):8384-92 (2003);Guiducci et al, J Immunol 172(7):4026-36 (2004)).

Targeted or gene trap mutations are generated in strain129SvEv^(Brd)-derived embryonic stem (ES) cells. The chimeric mice arebred to C57BL/6J albino mice to generate F1 heterozygous animals. Theseprogeny are intercrossed to generate F2 wild type, heterozygous, andhomozygous mutant progeny. On rare occasions, for example when very fewF1 mice are obtained from the chimera, F1 heterozygous mice are crossedto 129SvEv^(Brd)/C57 hybrid mice to yield additional heterozygousanimals for the intercross to generate the F2 mice. Level I phenotypicanalysis is performed on mice from this generation

wt het hom Total Observed 27 30 22 79 Expected 19.75 39.5 19.75 79Chi-Sq. = 0.5 Significance = 0.7788008 (hom/n) = 0.26 Avg. Litter Size =9Mutation InformationMutation Type Homologous Recombination (standard)Description: Coding exons 1 through 3 were targeted.1. Wild-type Expression Panel: Expression of the target gene wasdetected in embryonic stem (ES) cells and in brain, spinal cord, and eyeamong the 13 adult tissue samples tested by RT-PCR.2. QC Expression: QC Images: Disruption of the target gene was confirmedby Southern hybridization analysis.

48.27.1. Phenotypic Analysis (for Disrupted Gene: DNA82372 (UNQ886)

(a) Overall Phenotypic Summary:

Mutation of the gene encoding the ortholog of human chemokine (C-Cmotif) ligand 16 (CCL16) resulted in increased mean total tissue massand total body fat in female (−/−) mice. The mutant (−/−) mice alsoexhibited increased peritoneal CD117 cells and TCRb/CD38 cells inPeyer's patches. The mice showed an increased IL-6 response to the LPSchallenge as well as a decrease in mean serum IgG3 levels. Genedisruption was confirmed by Southern blot.

(b) Expression

HCC-4, also known as CCL-16, is a chemokine produced mainly in theliver. It is known to be a chemokine for monocytes and lymphocytes, butalso has a function in the angiogenic program in vascular endothelialcells. HCC-4 is upregulated in ulcerous colitis and is implicated ineosinophil trafficking via binding to the H4 histamine receptor.

(c) Immunology Phenotypic Analysis

Immune related and inflammatory diseases are the manifestation orconsequence of fairly complex, often multiple interconnected biologicalpathways which in normal physiology are critical to respond to insult orinjury, initiate repair from insult or injury, and mount innate andacquired defense against foreign organisms. Disease or pathology occurswhen these normal physiological pathways cause additional insult orinjury either as directly related to the intensity of the response, as aconsequence of abnormal regulation or excessive stimulation, as areaction to self, or as a combination of these.

Though the genesis of these diseases often involves multistep pathwaysand often multiple different biological systems/pathways, interventionat critical points in one or more of these pathways can have anameliorative or therapeutic effect. Therapeutic intervention can occurby either antagonism of a detrimental process/pathway or stimulation ofa beneficial process/pathway.

T lymphocytes (T cells) are an important component of a mammalian immuneresponse. T cells recognize antigens which are associated with aself-molecule encoded by genes within the major histocompatibilitycomplex (MHC). The antigen may be displayed together with MHC moleculeson the surface of antigen presenting cells, virus infected cells, cancercells, grafts, etc. The T cell system eliminates these altered cellswhich pose a health threat to the host mammal. T cells include helper Tcells and cytotoxic T cells. Helper T cells proliferate extensivelyfollowing recognition of an antigen-MHC complex on an antigen presentingcell. Helper T cells also secrete a variety of cytokines, i.e.,lymphokines, which play a central role in the activation of B cells,cytotoxic T cells and a variety of other cells which participate in theimmune response.

In many immune responses, inflammatory cells infiltrate the site ofinjury or infection. The migrating cells may be neutrophilic,eosinophilic, monocytic or lymphocytic as can be determined byhistological examination of the affected tissues. Current Protocols inImmunology, ed. John E. Coligan, 1994, John Wiley & Sons, Inc.

Many immune related diseases are known and have been extensivelystudied. Such diseases include immune-mediated inflammatory diseases(such as rheumatoid arthritis, immune mediated renal disease,hepatobiliary diseases, inflammatory bowel disease (IBD), psoriasis, andasthma), non-immune-mediated inflammatory diseases, infectious diseases,immunodeficiency diseases, neoplasia, and graft rejection, etc. In thearea of immunology, targets were identified for the treatment ofinflammation and inflammatory disorders.

In the area of immunology, targets have been identified herein for thetreatment of inflammation and inflammatory disorders. Immune relateddiseases, in one instance, could be treated by suppressing the immuneresponse. Using neutralizing antibodies that inhibit molecules havingimmune stimulatory activity would be beneficial in the treatment ofimmune-mediated and inflammatory diseases. Molecules which inhibit theimmune response can be utilized (proteins directly or via the use ofantibody agonists) to inhibit the immune response and thus ameliorateimmune related disease.

The following tests were performed:

Fluorescence-Activated Cell-Sorting (FACS) Analysis

Procedure:

FACS analysis of immune cell composition from peripheral blood wasperformed including CD4, CD8 and T cell receptor to evaluate Tlymphocytes, CD19 for B lymphocytes, CD45 as a leukocyte marker and panNK for natural killer cells. The FACS analysis was carried out on 2 wildtype and 6 homozygous mice and included cells derived from thymus,spleen, bone marrow and lymph node.

In these studies, analyzed cells were isolated from thymus, peripheralblood, spleen, bone marrow and lymph nodes. Flow cytometry was designedto determine the relative proportions of CD4 and CD8 positive T cells, Bcells, NK cells and monocytes in the mononuclear cell population. ABecton-Dickinson FACS Calibur 3-laser FACS machine was used to assessimmune status. For Phenotypic Assays and Screening, this machine recordsCD4+/CD8−, CD8+/CD4−, NK, B cell and monocyte numbers in addition to theCD4+/CD8+ ratio.

The mononuclear cell profile was derived by staining a single sample oflysed peripheral blood from each mouse with a panel of sixlineage-specific antibodies: CD45 PerCP, anti-TCRb APC, CD4 PE, CD8FITC, pan-NK PE, and CD19 FITC. The two FITC and PE labeled antibodiesstain mutually exclusive cell types. The samples were analyzed using aBecton Dickinson FACS Calibur flow cytometer with CellQuest software.

Results:

Tissue Specific FACS-Project: The (−/−) mice exhibited a phenotype inperitoneal CD117 cells (increase) and TCRb/CD38 cells in Peyer'spatches. Thus, PRO1904 polypeptides or the gene encoding PRO1904proteins appears to act as a negative regulator of hemopoieticprogenitor development and/or production.

Acute Phase Response:

Test Description: Bacterial lipopolysaccharide (LPS) is an endotoxin,and as such is a potent inducer of an acute phase response and systemicinflammation. The Level I LPS mice were injected intraperitoneally(i.p.) with a sublethal dose of LPS in 200 μL sterile saline using a 26gauge needle. The doses were based on the average weight of the micetested at 1 μg/g body weight 3 hours after injection; a 100 ul bloodsample was then taken and analyzed for the presence of TNFa, MCP-1, andIL-6 on the FACS Calibur instrument.

Results:

Acute Phase Response: The (−/−) mice exhibited an increased IL-6response to LPS challenge when compared with that of their (+/+)littermates and the historical mean.

In summary, the LPS endotoxin challenge demonstrated that knockout micedeficient in the gene encoding PRO1904 polypeptides exhibitimmunological abnormalities when compared with their wild-typelittermates. In particular, the mutant mice exhibited an increasedability to elicit an immunological response (IL-6 production) whenchallenged with the LPS endotoxin indicating a proinflammatory response.IL-6 contributes to the later stages of B cell activation. In addition,IL-6 plays a critical role in inducing the acute phase response andsystemic inflammation. This suggests that inhibitors or antagonists toPRO1904 polypeptides would stimulate the immune system and would findutility in the cases wherein this effect would be beneficial to theindividual such as in the case of leukemia, and other types of cancer,and in immuno-compromised patients, such as AIDS sufferers. Accordingly,PRO1904 polypeptides or agonists thereof would be useful in inhibitingthe immune response and would be useful candidates for suppressingharmful immune responses, e.g. in the case of graft rejection orgraft-versus-host diseases.

Serum Immunoglobulin Isotyping Assay:

The Serum Immunoglobulin Isotyping Assay is performed using a CytometricBead Array (CBA) kit. This assay is used to rapidly identify the heavyand light chain isotypes of a mouse monoclonal antibody in a singlesample. The values expressed are “relative fluorescence units” and arebased on the detection of kappa light chains. Any value <6 is notsignificant.

Results:

Serum Imm. 2: The (−/−) mice exhibited a decreased mean serum IgG3 levelwhen compared with those of their (+/+) littermates, the (+/+) micewithin the project run, and the historical medians for each.

The serum immunoglobulin isotyping assay showed decreased or reducedlevels of mean serum IgG3 in the homozygous (−/−) mice compared to theirgender-matched littermate (+/+) controls.

The serum immunoglobulin isotyping assay revealed that homozygous adultsexhibited decreased serum IgG3 levels. Thus, homozygotes showed anabnormally low serum immunoglobulins compared with the (+/+)littermates. Thus, the gene encoding PRO1904 is essential for makingimmunoglobulins (or gamma globulins). Likewise, Igg3 immunoglobulinshave neutralization effects and to a lesser extent are important foractivation of the complement system.

(d) Bone Metabolism & Body Diagnostics: Radiology Phenotypic Analysis

In the area of bone metabolism, targets were identified herein for thetreatment of arthritis, osteoporosis, osteopenia and osteopetrosis aswell as identifying targets that promote bone healing. Tests included:

DEXA for measurement of bone mineral density on femur and vertebra

MicroCT for very high resolution and very high sensitivity measurementsof bone mineral density for both trabecular and cortical bone.

Dexa Analysis—Test Description:

Procedure: A cohort of 4 wild type, 4 heterozygous and 8 homozygous micewere tested in this assay. Dual Energy X-ray Absorptiometry (DEXA) hasbeen used successfully to identify changes in bone. Anesthetized animalswere examined and bone mineral content (BMC), BMC/LBM ratios, volumetricbone mineral density (vBMD), total body BMD, femur BMD and vertebra BMDwere measured.

The mouse was anesthetized by intraperitoneal injection of Avertin(1.25% 2,2,2,-tribromoethanol, 20 ml/kg body weight), body length andweight were measured, and then the mouse was placed in a prone positionon the platform of the PIXImus™ Densitometer (Lunar Inc.) for a DEXAscan. Using Lunar PIXImus software, the bone mineral density (BMD) andfat composition (% fat) and total tissue mass (TTM) were determined inthe regions of interest (ROI) [i.e., whole body, vertebrae, and bothfemurs].

Bone MicroCT Analysis:

Procedure: MicroCT was also used to get very sensitive measurements ofBMD. One vertebra and 1 femur were taken from a cohort of 4 wild typeand 8 homozygous mice. Measurements were taken of lumbar 5 vertebratrabecular bone volume, trabecular thickness, connectivity density andmidshaft femur total bone area and cortical thickness. The μCT40 scansprovided detailed information on bone mass and architecture. Multiplebones were placed into sample holders and scanned automatically.Instrument software was used to select regions of interest for analysis.Trabecular bone parameters were analyzed in the fifth lumbar vertebrae(LV5) at 16 micrometer resolution and cortical bone parameters wereanalyzed in the femur midshaft at a resolution of 20 micrometers.

Results:

DEXA: The female (−/−) mice exhibited notably increased mean totaltissue mass, percent total body fat, and total fat mass when comparedwith that of their gender-matched (+/+) littermates and the historicalmeans. The female (−/−) mice also exhibited decreased mean total bodybone mineral content, bone mineral content index BMC/LBM, and vertebraebone mineral density (BMD). Increase in total tissue mass and total bodyfat with decreased bone-related measurements is significant since fatand bone cells arise from a common progenitor.

These studies suggest that mutant (−/−) non-human transgenic animalsexhibit a negative phenotype that would be associated with obesity.Thus, PRO1904 polypeptides or agonists thereof are essential for normalgrowth and metabolic processes and especially would be important in theprevention and/or treatment of obesity.

In addition, the decreased bone density and content measurements issuggestive of abnormal bone disorders. The (−/−) mice exhibited anegative bone phenotype with abnormal decreased bone measurementsreflective of bone metabolic disorders. The negative bone phenotypeindicates that PRO1904 polypeptides or agonists thereof would be usefulfor maintaining bone homeostasis. In addition, PRO1904 polypeptideswould be useful in bone healing or for the treatment of arthritis orosteoporosis, whereas antagonists (or inhibitors) of PRO1904polypeptides or its encoding gene would lead to abnormal or pathologicalbone disorders including inflammatory diseases associated with abnormalbone metabolism including arthritis, osteoporosis and osteopenia.

48.28. Generation and Analysis of Mice Comprising DNA225681 (UNQ983)Gene Disruptions

In these knockout experiments, the gene encoding PRO35193 polypeptides(designated as DNA225681) (UNQ983) was disrupted. The gene specificinformation for these studies is as follows: the mutated mouse genecorresponds to nucleotide reference: NM_(—)019447 ACCESSION:NM_(—)019447 NID:9506778 Mus musculus hepatocyte growth factor activator(Hgfac); protein reference: Q9R098ACCESSION: Q9R098NID: Mus musculus(Mouse). HEPATOCYTE GROWTH FACTOR ACTIVATOR PRECURSOR (EC 3.4.21.-) (HGFACTIVATOR) (HGFA); the human gene sequence reference: NM_(—)001528 Homosapiens HGF activator (HGFAC); the human protein sequence corresponds toreference: Q04756 ACCESSION: Q04756 NID: Homo sapiens (Human).HEPATOCYTE GROWTH FACTOR ACTIVATOR PRECURSOR (EC 3.4.21.-) (HGFACTIVATOR) (HGFA).

The mouse gene of interest is Hgfac (hepatocyte growth factoractivator), ortholog of human HGFAC. Aliases include HGFA.

HGFAC is a secreted protein expressed primarily in liver that functionsas a serine protease, cleaving and activating hepatocyte growth factor.The protein is expressed as an inactive zymogen that can be cleaved andactivated by thrombin. HGFAC is expressed not only in liver but also inureteric bud of the developing kidney and in multiple myeloma cells.HGFAC plays an important role in HGF signaling, which is involved indevelopment of the liver, kidney, placenta, lung, and mammary gland, inrepair of intestinal mucosa, and in growth and survival of multiplemyeloma cells (Itoh et al, Biochim Biophys Acta 1491(1-3):295-302(2000); Miyazawa et al, J Biol Chem 268(14): 10024-8 (1992); Shimomuraet al, J Biol Chem 268(30):22927-32 (1993); van Adelsberg et al, J BiolChem 276(18):15099-106 (2001); Itoh et al, Gastroenterology127(5):1423-35 (2004); Tjin et al, Blood 104(7):2172-5 (2004).

Itoh and colleagues (2004) investigated the physiological role of HGFACin knockout mice. They showed that death resulting from gastrointestinalinjury by oral administration of dextran sodium sulfate was higher inHGFAC homozygous null mice than in wild-type mice. Moreover, they showedthat HGF activation and repair of injured mucosa by regeneratingepithelium was impaired in homozygous null mice but not in wild-typemice. Itoh and colleagues concluded that HGFAC is required for repair ofinjured intestinal mucosa but is not essential for normal development.

Targeted or gene trap mutations are generated in strain129SvEv^(Brd)-derived embryonic stem (ES) cells. The chimeric mice arebred to C57BL/6J albino mice to generate F1 heterozygous animals. Theseprogeny are intercrossed to generate F2 wild type, heterozygous, andhomozygous mutant progeny. On rare occasions, for example when very fewF1 mice are obtained from the chimera, F1 heterozygous mice are crossedto 129SvEv^(Brd)/C57 hybrid mice to yield additional heterozygousanimals for the intercross to generate the F2 mice. Level I phenotypicanalysis is performed on mice from this generation

wt het hom Total Observed 24 44 20 88 Expected 22 44 22 88 Chi-Sq. =0.03 Significance = 0.98511195 (hom/n) = 0.25 Avg. Litter Size = 9Mutation InformationMutation Type: Homologous Recombination (standard)Description: Coding exons 1 through 9 were targeted (NCBI accessionNM_(—)019447.1).1. Wild-type Expression Panel: Expression of the target gene wasdetected in embryonic stem (ES) cells and in all 13 adult tissue samplestested by RT-PCR, except skeletal muscle and bone.2. QC Expression: QC Images: Disruption of the target gene was confirmedby Southern hybridization analysis.

48.28.1. Phenotypic Analysis (for Disrupted Gene: DNA225681 (UNQ983)

(a) Overall Phenotypic Summary:

Mutation of the gene encoding the ortholog of human hepatocyte growthfactor activator (HGFAC) resulted in the (−/−) mice exhibiting increasedmean serum glucose levels. Gene disruption was confirmed by Southernblot.

(b) Phenotypic Analysis: Metabolism—Blood Chemistry

In the area of metabolism, targets may be identified for the treatmentof diabetes. Blood chemistry phenotypic analysis includes blood glucosemeasurements. The COBAS Integra 400 (mfr: Roche) was used for runningblood chemistry tests on the mice. In the area of metabolism, targetsmay be identified for the treatment of diabetes.

Results:

The female (−/−) mice exhibited an increased mean serum glucose levelwhich could be related to abnormal glucose metabolism and/or diabetes.

Thus, the mutant (−/−) mice exhibited hyperglycemia which could beassociated with an altered glucose metabolism or diabetes. PRO35193polypeptides or agonists thereof would be useful in maintaining normalglucose levels/metabolism and possibly useful in the treatment ofdiabetes.

48.29. Generation and Analysis of Mice Comprising DNA81761-2583(UNQ1895) Gene Disruptions

In these knockout experiments, the gene encoding PRO4341 polypeptides(designated as DNA81761-2583) (UNQ1895) was disrupted. The gene specificinformation for these studies is as follows: the mutated mouse genecorresponds to nucleotide reference: NM_(—)019454 ACCESSION:NM_(—)019454NID:9506546 Mus musculus Mus musculus delta-like 4(Drosophila) (D114); protein reference: Q9 DBU9 ACCESSION: Q9 DBU9 NID:Mus musculus (Mouse). DELTA-LIKE 4 HOMOLOG (DROSOPHILA); the human genesequence reference: NM_(—)019074 Homo sapiens delta-like 4 (Drosophila)(DLL4); the human protein sequence corresponds to reference: Q9NR61ACCESSION: Q9NR61 NID: Homo sapiens (Human). DELTA-LIKE PROTEIN 4PRECURSOR (DROSOPHILA DELTA HOMOLOG 4).

The mouse gene of interest is D114 (delta-like 4 [Drosophila]), orthologof human DLL4. Aliases include Delta4, delta-like 4 protein, delta 4precursor, delta ligand 4 precursor, notch ligand DLL4 precursor, notchligand delta-2 precursor, delta-like 4 homolog (Drosophila), andhdelta2.

DLL4 is a type I plasma membrane protein belonging to the Delta familyof Notch ligands. DLL4 contains a signal peptide, a delta serrate ligand(DSL) domain, at least seven epidermal growth factor (EGF)-like repeats,a transmembrane segment, and a cytoplasmic C-terminus. DLL4 is capableof activating receptors NOTCH 1 and NOTCH4 (Shutter et al, Genes Dev14(11):1313-8 (2000)), which play an important role in angiogenesis(Krebs et al, Genes Dev 14(11):1343-52 (2000)). DLL4 is expressedprimarily in vascular endothelium of arteries in the developing mouseembryo, in adult mice, and in tumor models (Shutter et al, Genes Dev14(11):1313-8 (2000); Mailhos et al, Differentiation 69(2-3):135-44(2001)). DLL4 plays a role in hematopoietic and vascular development(Dorsch et al, Blood 100(6):2046-55 (2002); Lauret et al, Leukemia18(4):788-97 (2004)) and may have therapeutic potential for treatment ofcertain types of cancer (Tohda et al, Int J Oncol 22(5): 1073-9 (2003)).

Several investigators have studied the physiological role of DLL4 usingknockout mice. Krebs and coworkers [Genes Dev 18(20):2469-73 (2004)] aswell as Gale and colleagues [Proc Natl Acad Sci USA 101(45):15949-54(2004)] showed that vascular remodeling was defective in DLL4heterozygous embryos, resulting in haplo-insufficient lethality. Theyconcluded that vascular remodeling is sensitive to DLL4 gene dosage.Duarte and colleagues [Genes Dev 18(20):2474-8 (2004)] successfullygenerated DLL4 heterozygous mice by crossing germ line transmittingchimeras with ICR female mice. DLL4 heterozygous mice were produced with27% frequency in the ICR background, whereas no DLL4 heterozygous micewere produced in a 129/Sv-CP background. Defects in arterial vasculardevelopment were evident to varying degrees in all DLL4 heterozygousembryos. Surviving male and female DLL4 heterozygous mice wereapparently normal and fertile. Embryonic lethality was observed at day10.5 in DLL4 homozygous null mice, showing severe defects primarily inarterial vascular development. Duarte and colleagues concluded thatembryonic vascular development is very sensitive to DLL4 levels asevidenced in part by strain-dependent haplo-insufficiency. Theysuggested that DLL4 may have therapeutic utility for intervention inadult neovascularization.

Targeted or gene trap mutations are generated in strain129SvEv^(Brd)-derived embryonic stem (ES) cells. The chimeric mice arebred to C57BL/6J albino mice to generate F1 heterozygous animals. Theseprogeny are intercrossed to generate F2 wild type, heterozygous, andhomozygous mutant progeny. On rare occasions, for example when very fewF1 mice are obtained from the chimera, F1 heterozygous mice are crossedto 129SvEv^(Brd)/C57 hybrid mice to yield additional heterozygousanimals for the intercross to generate the F2 mice. Level I phenotypicanalysis is performed on mice from this generation

wt het hom Total Observed 0 0 0 0 Expected 0 0 0 0 Chi-Sq. = 0.0Significance = 0.0 (hom/n) = 0.0 Avg. Litter Size = 0Mutation InformationMutation Type: Homologous Recombination (standard)Description: Coding exons 1 through 8 were targeted (NCBI accessionNM_(—)019454.1).1. Wild-type Expression Panel: Expression of the target gene wasdetected in all 26 adult tissue samples tested by RT-PCR, exceptskeletal muscle, asthmatic lung, LPS liver, blood, skin fibroblast, MG12 DPC, and MG 3 day post-weaning (lactating).2. QC Expression: QC Images: Disruption of the target gene was confirmedby Southern hybridization analysis.

48.29.1. Phenotypic Analysis (for Disrupted Gene: DNA81761-1583(UNQ1895)

(a) Overall Phenotypic Summary:

Mutation of the gene encoding the ortholog of human delta-like 4(Drosophila) (DLL4) resulted in lethality of (+/−) and (−/−) mutants.Genetic data indicate that this mutation resulted in lethality of boththe heterozygous and homozygous mutants. There were no structuraldevelopmental abnormalities detected in the heterozygous embryosexamined at 11.5 and 12.5 days. No homozygous mutant embryos wereobserved.

Disruption of the target gene was confirmed by Southern hybridizationanalysis.

(b) Pathology

Microscopic: Embryonic lethal. No (−/−) embryos were observed. Therewere no structural developmental abnormalities detected in the 11.5 dayor 12.5 day (+/−) embryos.

Discussion Related to Embryonic Developmental Abnormality of Lethality:

Embryonic lethality in knockout mice usually results from variousserious developmental problems including but not limited toneuro-degenerative diseases, angiogenic disorders, inflammatorydiseases, or where the gene/protein has an important role in basic cellsignaling processes in many cell types. In addition, embryonic lethalsare useful as potential cancer models. Likewise, the correspondingheterozygous (+/−) mutant animals are particularly useful when theyexhibit a phenotype and/or a pathology report which reveals highlyinformative clues as to the function of the knocked-out gene. Forinstance, EPO knockout animals were embryonic lethals, but the pathologyreports on the embryos showed a profound lack of RBCs.

48.30. Generation and Analysis of Mice Comprising DNA92232-2589(UNQ1902) Gene Disruptions

In these knockout experiments, the gene encoding PRO4348 polypeptides(designated as DNA92232-2589) (UNQ1902) was disrupted. The gene specificinformation for these studies is as follows: the mutated mouse genecorresponds to nucleotide reference: NM_(—)027334 ACCESSION:NM_(—)027334 NID: gi 33563289 ref NM_(—)027334.2 Mus musculus RIKEN cDNA3300001H21 gene (3300001H21Rik); protein reference: Q8C6B0 ACCESSION:Q8C6B0 NID: Mus musculus (Mouse). Mus musculus 17 days embryo head cDNA,RIKEN full-length enriched library, clone:3300001H21product:hypothetical S-adenosyl-L-methionine-dependentmethyltransferases structure containing protein, full insert sequence(DKFZP586A0522 protein); the human gene sequence reference: NM_(—)014033Homo sapiens DKFZP586A0522 protein (DKFZP586A0522); the human proteinsequence corresponds to reference: Q9H8H3 ACCESSION: Q9H8H₃NID: Homosapiens (Human). cDNA FLJ13631 FIS, CLONE PLACE1011090, HIGHLY SIMILARTO HOMO SAPIENS mRNA; cDNA DKFZP586A0522 (FROM CLONE DKFZP586A0522)(UNKNOWN) (PROTEIN FOR MGC:11081) (DKFZP586A0522 PROTEIN).

The mouse gene of interest is RIKEN cDNA 3300001H21 gene, ortholog ofhuman DKFZP586A0522 protein. Aliases include 2210414H16Rik, UbiE, Aam-B,and AAM-B protein.

DKFZP586A0522 protein is a putative type II integral membrane proteinthat may function as a methyltransferase enzyme. The protein contains asignal anchor and an S-adenosyl-L-methionine-dependentmethyltransferases superfamily domain (SCOP accession dlfp2a2; InterProaccessions IPR000051 and IPR001601). Proteins with this domain catalyzethe methylation of specific DNA, RNA, proteins, or small moleculesubstrates, using S-adenosyl-L-methionine as the methyl donor.DKFZP586A0522 protein may be an extracellular protein (Clark et al,Genome Res 13(10):2265-70 (2003)).

Targeted or gene trap mutations are generated in strain129SvEv^(Brd)-derived embryonic stem (ES) cells. The chimeric mice arebred to C57BL/6J albino mice to generate F1 heterozygous animals. Theseprogeny are intercrossed to generate F2 wild type, heterozygous, andhomozygous mutant progeny. On rare occasions, for example when very fewF1 mice are obtained from the chimera, F1 heterozygous mice are crossedto 129SvEv^(Brd)/C57 hybrid mice to yield additional heterozygousanimals for the intercross to generate the F2 mice. Level I phenotypicanalysis is performed on mice from this generation

wt het hom Total Observed 24 40 19 83 Expected 20.75 41.5 20.75 83Chi-Sq. = 2.3 Significance = 0.31663677 (hom/n) = 0.23 Avg. Litter Size= 9Mutation InformationMutation Type Homologous Recombination (standard)Description: Coding exon 1 was targeted (NCBI accession NM_(—)027334).1. Wild-type Expression Panel: Expression of the target gene wasdetected in embryonic stem (ES) cells and in all 26 adult tissue samplestested by RT-PCR.2. QC Expression: QC Images: Disruption of the target gene was confirmedby Southern hybridization analysis.

48.30.1. Phenotypic Analysis (for Disrupted Gene: DNA92232-2589(UNQ1902)

(a) Overall Phenotypic Summary:

Mutation of the gene encoding the ortholog of a putative human type IIintegral membrane protein (DKFZP586A0522) resulted in the mutant (−/−)mice exhibiting decreased bone mineral content and bone mineral densitymeasurements. Gene disruption was confirmed by Southern blot.

(b) Bone Metabolism & Body Diagnostics: Radiology Phenotypic Analysis

In the area of bone metabolism, targets were identified herein for thetreatment of arthritis, osteoporosis, osteopenia and osteopetrosis aswell as identifying targets that promote bone healing. Tests included:

DEXA for measurement of bone mineral density on femur and vertebra

MicroCT for very high resolution and very high sensitivity measurementsof bone mineral density for both trabecular and cortical bone.

Dexa Analysis—Test Description:

Procedure: A cohort of 4 wild type, 4 heterozygous and 8 homozygous micewere tested in this assay. Dual Energy X-ray Absorptiometry (DEXA) hasbeen used successfully to identify changes in bone. Anesthetized animalswere examined and bone mineral content (BMC), BMC/LBM ratios, volumetricbone mineral density (vBMD), total body BMD, femur BMD and vertebra BMDwere measured.

The mouse was anesthetized by intraperitoneal injection of Avertin(1.25% 2,2,2,-tribromoethanol, 20 ml/kg body weight), body length andweight were measured, and then the mouse was placed in a prone positionon the platform of the PIXImus™ Densitometer (Lunar Inc.) for a DEXAscan. Using Lunar PIXImus software, the bone mineral density (BMD) andfat composition (% fat) and total tissue mass (TTM) were determined inthe regions of interest (ROI) [i.e., whole body, vertebrae, and bothfemurs].

Bone MicroCT Analysis:

Procedure: MicroCT was also used to get very sensitive measurements ofBMD. One vertebra and 1 femur were taken from a cohort of 4 wild typeand 8 homozygous mice. Measurements were taken of lumbar 5 vertebratrabecular bone volume, trabecular thickness, connectivity density andmidshaft femur total bone area and cortical thickness. The μCT40 scansprovided detailed information on bone mass and architecture. Multiplebones were placed into sample holders and scanned automatically.Instrument software was used to select regions of interest for analysis.Trabecular bone parameters were analyzed in the fifth lumbar vertebrae(LV5) at 16 micrometer resolution and cortical bone parameters wereanalyzed in the femur midshaft at a resolution of 20 micrometers.

Results:

DEXA: The female (−/−) mice exhibited decreased mean bone mineralcontent, total body bone mineral density, and vertebrae bone mineraldensity measurements when compared with those of their gender-matched(+/+) littermates and the historical means.

The decreased bone density and content measurements is suggestive ofabnormal bone disorders. The (−/−) mice exhibited a negative bonephenotype with abnormal decreased bone measurements reflective of bonemetabolic disorders. The negative bone phenotype indicates that PRO4348polypeptides or agonists thereof would be useful for maintaining bonehomeostasis. In addition, PRO4348 polypeptides would be useful in bonehealing or for the treatment of arthritis or osteoporosis, whereasantagonists (or inhibitors) of PRO4348 polypeptides or its encoding genewould lead to abnormal or pathological bone disorders includinginflammatory diseases associated with abnormal bone metabolism includingarthritis, osteoporosis and osteopenia.

48.31. Generation and Analysis of Mice Comprising DNA92289-2598(UNQ1911) Gene Disruptions

In these knockout experiments, the gene encoding PRO4369 polypeptides(designated as DNA92289-2598) (UNQ1911) was disrupted. The gene specificinformation for these studies is as follows: the mutated mouse genecorresponds to nucleotide reference: NM_(—)023403 ACCESSION:NM_(—)023403 NID: gi 12963664 ref NM_(—)023403.1 Mus musculus mesodermdevelopment candidate 2 (Mesdc2); protein reference: Q9ERE7 ACCESSION:Q9ERE7 NID: Mus musculus (Mouse). Mesoderm development candidate 2; thehuman gene sequence reference: BC009210 ACCESSION:BC009210 NID:14327971Homo sapiens, Similar to mesoderm development candidate 2, cloneMGC:16185 IMAGE:3637449; the human protein sequence corresponds toreference: Q14696 ACCESSION: Q14696 NID: Homo sapiens (Human). Mesodermdevelopment candidate 2.

The mouse gene of interest is Mesdc2 (mesoderm development candidate 2),ortholog of human MESDC2. Aliases include MGC25959, mKIAA0081,2210015O1Rik, BOCA, MESD, and KIAA0081.

MESDC2 is a protein located on the endoplasmic reticulum that likelyfunctions as a chaperone protein for Wnt signaling coreceptors LRP5 andLRP6 or for other low-density lipoprotein receptor family members.MESDC2 may play a role in processes involving cargo transport or Wntsignaling, such as embryonic polarity and mesoderm induction duringdevelopment and bone formation (Wines et al, Genomics 72(1):88-98(2001); Hsieh et al, Cell 112(3):355-67 (2003); Culi and Mann, Cell112(3):343-54 (2003); Herz and Marschang, Cell 112(3):289-92 (2003);Zhang et al, Mol Cell Biol 24(11):4677-84 (2004)).

Targeted or gene trap mutations are generated in strain129SvEv^(Brd)-derived embryonic stem (ES) cells. The chimeric mice arebred to C57BL/6J albino mice to generate F1 heterozygous animals. Theseprogeny are intercrossed to generate F2 wild type, heterozygous, andhomozygous mutant progeny. On rare occasions, for example when very fewF1 mice are obtained from the chimera, F1 heterozygous mice are crossedto 129SvEv^(Brd)/C57 hybrid mice to yield additional heterozygousanimals for the intercross to generate the F2 mice. Level I phenotypicanalysis is performed on mice from this generation

wt het hom Total Observed 21 32 0 53 Expected 13.25 26.5 13.25 53Chi-Sq. = 42.69 Significance = 5.370127E−10 (hom/n) = 0.0 Avg. LitterSize = 8Mutation InformationMutation Type Homologous Recombination (standard)Description: Coding exon 1 was targeted (NCBI accession NM_(—)023403.2).1. Wild-type Expression Panel: Expression of the target gene wasdetected in embryonic stem (ES) cells and in all 13 adult tissue samplestested by RT-PCR.2. QC Expression: QC Images: Disruption of the target gene was confirmedby Southern hybridization analysis.

48.31.1. Phenotypic Analysis (for Disrupted Gene: DNA92289-2598(UNQ1911)

(a) Overall Phenotypic Summary:

Mutation of the UNQ1911 gene encoding the ortholog of human mesodermdevelopment candidate 2 (MESDC2) resulted in lethality of (−/−) mutants.Genetic data indicate that this mutation resulted in lethality of thehomozygous mutants. The heterozygous mice exhibited increased mean serumIgG1 and IgG2a responses to ovalbumin challenge when compared with thoseof their wild-type littermates and the historical means. Increased meanserum IgM levels were also observed in the (+/−) mice. Disruption of thetarget gene was confirmed by Southern hybridization analysis.

(b) Pathology

Microscopic: Due to embryonic lethality, microscopic analysis was notperformed. At 12.5 days, there were 47 embryos observed: 27 (+/−)embryos, 4 (+/+) embryos, 14 resorption moles, and 2 inconclusive.UNQ1911 is a protein of unknown function that lies within a chromosomalregion critical for the differentiation of mesoderm. UNQ1911 is likelyto play a key role in regulating mesoderm differentiation which couldexplain the resultant embryonic lethality in the homozygous mice. GeneExpression: LacZ activity was not detected in the panel of tissues byimmuno-histochemical analysis.

Discussion Related to Embryonic Developmental Abnormality of Lethality:

Embryonic lethality in knockout mice usually results from variousserious developmental problems including but not limited toneuro-degenerative diseases, angiogenic disorders, inflammatorydiseases, or where the gene/protein has an important role in basic cellsignaling processes in many cell types. In addition, embryonic lethalsare useful as potential cancer models. Likewise, the correspondingheterozygous (+/−) mutant animals are particularly useful when theyexhibit a phenotype and/or a pathology report which reveals highlyinformative clues as to the function of the knocked-out gene. Forinstance, EPO knockout animals were embryonic lethals, but the pathologyreports on the embryos showed a profound lack of RBCs.

(c) Immunology Phenotypic Analysis

Immune related and inflammatory diseases are the manifestation orconsequence of fairly complex, often multiple interconnected biologicalpathways which in normal physiology are critical to respond to insult orinjury, initiate repair from insult or injury, and mount innate andacquired defense against foreign organisms. Disease or pathology occurswhen these normal physiological pathways cause additional insult orinjury either as directly related to the intensity of the response, as aconsequence of abnormal regulation or excessive stimulation, as areaction to self, or as a combination of these.

Though the genesis of these diseases often involves multistep pathwaysand often multiple different biological systems/pathways, interventionat critical points in one or more of these pathways can have anameliorative or therapeutic effect. Therapeutic intervention can occurby either antagonism of a detrimental process/pathway or stimulation ofa beneficial process/pathway.

T lymphocytes (T cells) are an important component of a mammalian immuneresponse. T cells recognize antigens which are associated with aself-molecule encoded by genes within the major histocompatibilitycomplex (MHC). The antigen may be displayed together with MHC moleculeson the surface of antigen presenting cells, virus infected cells, cancercells, grafts, etc. The T cell system eliminates these altered cellswhich pose a health threat to the host mammal. T cells include helper Tcells and cytotoxic T cells. Helper T cells proliferate extensivelyfollowing recognition of an antigen-MHC complex on an antigen presentingcell. Helper T cells also secrete a variety of cytokines, i.e.,lymphokines, which play a central role in the activation of B cells,cytotoxic T cells and a variety of other cells which participate in theimmune response.

In many immune responses, inflammatory cells infiltrate the site ofinjury or infection. The migrating cells may be neutrophilic,eosinophilic, monocytic or lymphocytic as can be determined byhistological examination of the affected tissues. Current Protocols inImmunology, ed. John E. Coligan, 1994, John Wiley & Sons, Inc.

Many immune related diseases are known and have been extensivelystudied. Such diseases include immune-mediated inflammatory diseases(such as rheumatoid arthritis, immune mediated renal disease,hepatobiliary diseases, inflammatory bowel disease (IBD), psoriasis, andasthma), non-immune-mediated inflammatory diseases, infectious diseases,immunodeficiency diseases, neoplasia, and graft rejection, etc. In thearea of immunology, targets were identified for the treatment ofinflammation and inflammatory disorders.

In the area of immunology, targets have been identified herein for thetreatment of inflammation and inflammatory disorders. Immune relateddiseases, in one instance, could be treated by suppressing the immuneresponse. Using neutralizing antibodies that inhibit molecules havingimmune stimulatory activity would be beneficial in the treatment ofimmune-mediated and inflammatory diseases. Molecules which inhibit theimmune response can be utilized (proteins directly or via the use ofantibody agonists) to inhibit the immune response and thus ameliorateimmune related disease.

The following tests were performed:

Ovalbumin Challenge

Procedure: This assay was carried out on 7 wild types and 8 heterozygousmice. Chicken ovalbumin (OVA) is a T-cell dependent antigen, which iscommonly used as a model protein for studying antigen-specific immuneresponses in mice. OVA is non-toxic and inert and therefore will notcause harm to the animals even if no immune response is induced. Themurine immune response to OVA has been well characterized, to the extentthat the immuno-dominant peptides for eliciting T cell responses havebeen identified. Anti-OVA antibodies are detectable 8 to 10 days afterimmunization using enzyme-linked immunosorbent assay (ELIZA), anddetermination of different isotypes of antibodies gives furtherinformation on the complex processes that may lead to a deficientresponse in genetically engineered mice.

As noted above, this protocol assesses the ability of mice to raise anantigen-specific immune response. Animals were injected IP with 50 mg ofchicken ovalbumin emulsified in Complete Freund's Adjuvant and 14 dayslater the serum titer of anti-ovalbumin antibodies (IgM, IgG1 and IgG2subclasses) was measured. The amount of OVA-specific antibody in theserum sample is proportional to the Optical Density (OD) value generatedby an instrument that scans a 96-well sample plate. Data was collectedfor a set of serial dilutions of each serum sample.

Results of this Challenge:

Ovalbumin: The (+/−) mice exhibited increased mean serum IgG1 and IgG2aresponses to ovalbumin challenge when compared with those of their (+/+)littermates and the historical means.

In summary, the ovalbumin challenge studies indicate that knockoutheterozygous mice deficient in the gene encoding PRO4348 polypeptidesexhibit immunological abnormalities when compared with their wild-typelittermates. In particular, the mutant (+/−) mice exhibited an increasedability to elicit an immunological response when challenged with theT-cell dependent OVA antigen. Thus, antagonists (inhibitors) of PRO4348polypeptides would be useful for stimulating the immune system (such asT cell proliferation) and would find utility in the cases wherein thiseffect would be beneficial to the individual such as in the case ofleukemia, and other types of cancer, and in immuno-compromised patients,such as AIDS sufferers. Accordingly, PRO4348 polypeptides or agoniststhereof, would be useful for inhibiting the immune response and thuswould be useful candidates for suppressing harmful immune responses,e.g. in the case of graft rejection or graft-versus-host diseases.

Serum Immunoglobulin Isotyping Assay:

The Serum Immunoglobulin Isotyping Assay is performed using a CytometricBead Array (CBA) kit. This assay is used to rapidly identify the heavyand light chain isotypes of a mouse monoclonal antibody in a singlesample. The values expressed are “relative fluorescence units” and arebased on the detection of kappa light chains. Any value <6 is notsignificant.

Results:

Serum Imm. 2: The (+/−) mice exhibited an increased mean serum IgM levelwhen compared with that of their (+/+) littermates, the (+/+) micewithin the project run, and the historical range.

Mutant (+/−) mice exhibited elevation of IgM serum immunoglobulinscompared to their gender-matched (+/+) littermates. IgM immunoglobulinsare the first to be produced in a humoral immune response forneutralization of bacterial toxins and are particularly important inactivating the complement system. The observed phenotype suggests thatthe PRO4348 polypeptide is a negative regulator of inflammatoryresponses. These immunological abnormalities suggest that inhibitors(antagonists) of PRO4348 polypeptides would be useful in stimulating theimmune system (such as T cell proliferation) and would find utility inthe cases wherein this effect would be beneficial to the individual suchas in the case of leukemia, and other types of cancer, and inimmunocompromised patients, such as AIDS sufferers. Accordingly, PRO4348polypeptides or agonists thereof would be useful in inhibiting theimmune response and would be useful candidates for suppressing harmfulimmune responses, e.g. in the case of graft rejection orgraft-versus-host diseases.

48.32. Generation and Analysis of Mice Comprising DNA92225-2603(UNQ1916) Gene Disruptions

In these knockout experiments, the gene encoding PRO4381 polypeptides(designated as DNA92225-2603) (UNQ1916) was disrupted. The gene specificinformation for these studies is as follows: the mutated mouse genecorresponds to nucleotide reference: NM_(—)178066 Mus musculus RIKENcDNA 1110012D08 gene (1110012D08Rik); protein reference: Q8CFU0ACCESSION: Q8CFU0 NID: Mus musculus (Mouse). RIKEN cDNA 1110012D08; thehuman gene sequence reference: AK057179 ACCESSION:AK057179 NID:16552774Homo sapiens cDNA FLJ32617 fis, clone STOMA2000257.

The mouse gene of interest is RIKEN cDNA 1110012D08 gene, which isorthologous with a human gene represented by “Homo sapiens cDNA FLJ32617fis, clone STOMA2000257.”

The hypothetical protein is a likely integral membrane protein,consisting of seven transmembrane domains. Bioinformatic analysissuggests that the hypothetical protein is located on the plasmamembrane.

Targeted or gene trap mutations are generated in strain129SvEv^(Brd)-derived embryonic stem (ES) cells. The chimeric mice arebred to C57BL/6J albino mice to generate F1 heterozygous animals. Theseprogeny are intercrossed to generate F2 wild type, heterozygous, andhomozygous mutant progeny. On rare occasions, for example when very fewF1 mice are obtained from the chimera, F1 heterozygous mice are crossedto 129SvEv^(Brd)/C57 hybrid mice to yield additional heterozygousanimals for the intercross to generate the F2 mice. Level I phenotypicanalysis is performed on mice from this generation

wt het hom Total Observed 20 41 20 81 Expected 20.25 40.5 20.25 81Chi-Sq. = 0.83 Significance = 0.6603403 (hom/n) = 0.27 Avg. Litter Size= 9Mutation InformationMutation Type: Homologous Recombination (standard)Description: The noncoding exon preceding coding exon 1 and coding exons1 and 2 were targeted (NCBI accession NM_(—)178066.2).1. Wild-type Expression Panel: Expression of the target gene wasdetected in embryonic stem (ES) cells and in all 13 adult tissue samplestested by RT-PCR, except bone.2. QC Expression: QC Images: Disruption of the target gene was confirmedby Southern hybridization analysis.

48.32.1. Phenotypic Analysis (for Disrupted Gene: DNA92225-2603(UNQ1916)

(a) Overall Phenotypic Summary:

Mutation of the gene encoding the ortholog of a human hypotheticalmembrane protein resulted in impaired sensorimotor gating/attention in(−/−) mice. The male (−/−) mice exhibited increased mean body weightwhen compared with that of their gender-matched (+/+) littermates andthe historical mean. Immunological abnormalities were also observed inthe (−/−) mice since the homozygous mice exhibited increased plateletcounts and decreased mean percentage of CD8 cells compared to their(+/+) littermate controls. Gene disruption was confirmed by Southernblot.

(b) Immunology Phenotypic Analysis

Immune related and inflammatory diseases are the manifestation orconsequence of fairly complex, often multiple interconnected biologicalpathways which in normal physiology are critical to respond to insult orinjury, initiate repair from insult or injury, and mount innate andacquired defense against foreign organisms. Disease or pathology occurswhen these normal physiological pathways cause additional insult orinjury either as directly related to the intensity of the response, as aconsequence of abnormal regulation or excessive stimulation, as areaction to self, or as a combination of these.

Though the genesis of these diseases often involves multistep pathwaysand often multiple different biological systems/pathways, interventionat critical points in one or more of these pathways can have anameliorative or therapeutic effect. Therapeutic intervention can occurby either antagonism of a detrimental process/pathway or stimulation ofa beneficial process/pathway.

T lymphocytes (T cells) are an important component of a mammalian immuneresponse. T cells recognize antigens which are associated with aself-molecule encoded by genes within the major histocompatibilitycomplex (MHC). The antigen may be displayed together with MHC moleculeson the surface of antigen presenting cells, virus infected cells, cancercells, grafts, etc. The T cell system eliminates these altered cellswhich pose a health threat to the host mammal. T cells include helper Tcells and cytotoxic T cells. Helper T cells proliferate extensivelyfollowing recognition of an antigen-MHC complex on an antigen presentingcell. Helper T cells also secrete a variety of cytokines, i.e.,lymphokines, which play a central role in the activation of B cells,cytotoxic T cells and a variety of other cells which participate in theimmune response.

In many immune responses, inflammatory cells infiltrate the site ofinjury or infection. The migrating cells may be neutrophilic,eosinophilic, monocytic or lymphocytic as can be determined byhistological examination of the affected tissues. Current Protocols inImmunology, ed. John E. Coligan, 1994, John Wiley & Sons, Inc.

Many immune related diseases are known and have been extensivelystudied. Such diseases include immune-mediated inflammatory diseases(such as rheumatoid arthritis, immune mediated renal disease,hepatobiliary diseases, inflammatory bowel disease (IBD), psoriasis, andasthma), non-immune-mediated inflammatory diseases, infectious diseases,immunodeficiency diseases, neoplasia, and graft rejection, etc. In thearea of immunology, targets were identified for the treatment ofinflammation and inflammatory disorders.

In the area of immunology, targets have been identified herein for thetreatment of inflammation and inflammatory disorders. Immune relateddiseases, in one instance, could be treated by suppressing the immuneresponse. Using neutralizing antibodies that inhibit molecules havingimmune stimulatory activity would be beneficial in the treatment ofimmune-mediated and inflammatory diseases. Molecules which inhibit theimmune response can be utilized (proteins directly or via the use ofantibody agonists) to inhibit the immune response and thus ameliorateimmune related disease.

The following tests were performed:

Fluorescence-Activated Cell-Sorting (FACS) Analysis

Procedure:

FACS analysis of immune cell composition from peripheral blood wasperformed including CD4, CD8 and T cell receptor to evaluate Tlymphocytes, CD19 for B lymphocytes, CD45 as a leukocyte marker and panNK for natural killer cells. The FACS analysis was carried out on 2 wildtype and 6 homozygous mice and included cells derived from thymus,spleen, bone marrow and lymph node.

In these studies, analyzed cells were isolated from thymus, peripheralblood, spleen, bone marrow and lymph nodes. Flow cytometry was designedto determine the relative proportions of CD4 and CD8 positive T cells, Bcells, NK cells and monocytes in the mononuclear cell population. ABecton-Dickinson FACS Calibur 3-laser FACS machine was used to assessimmune status. For Phenotypic Assays and Screening, this machine recordsCD4+/CD8−, CD8+/CD4−, NK, B cell and monocyte numbers in addition to theCD4+/CD8+ ratio.

The mononuclear cell profile was derived by staining a single sample oflysed peripheral blood from each mouse with a panel of sixlineage-specific antibodies: CD45 PerCP, anti-TCRb APC, CD4 PE, CD8FITC, pan-NK PE, and CD19 FITC. The two FITC and PE labeled antibodiesstain mutually exclusive cell types. The samples were analyzed using aBecton Dickinson FACS Calibur flow cytometer with CellQuest software.

Results:

FACS3: The (−/−) mice exhibited an altered distribution of leukocytesubsets in the peripheral blood, characterized by a decreased meanpercentage of CD8 cells when compared with that of their (+/+)littermates and the historical mean.

Thus, knocking out the gene which encodes PRO4381 polypeptides causes adecrease in the T cell population. From these observations, PRO4381polypeptides or the gene encoding PRO4381 appears to act as a positiveregulator of T cell proliferation. Thus, PRO4381 polypeptides would bebeneficial in enhancing T cell proliferation.

Hematology Analysis:

Test Description: Blood tests are carried out by Abbott's Cell-Dyn3500R, an automated hematology analyzer. Some of its features include afive-part WBC differential. ‘Patient’ reports can cover over 22parameters in all.

Results:

Hematology: The (−/−) mice exhibited an increased mean platelet countwhen compared with their (+/+) littermates and the historical mean.

Thus, mutant mice deficient in the DNA92225-2603 gene resulted in aphenotype related to coagulation disorders. In this regard, inhibitorsor antagonists of PRO4381 polypeptides would be useful in treatingdisorders related to abnormal blood coagulation such as hemophilia.

(c) Phenotypic Analysis: CNS/Neurology

In the area of neurology, analysis focused herein on identifying in vivovalidated targets for the treatment of neurological and psychiatricdisorders including depression, generalized anxiety disorders, attentiondeficit hyperactivity disorder, obsessive compulsive disorder,schizophrenia, cognitive disorders, hyperalgesia and sensory disorders.Neurological disorders include the category defined as “anxietydisorders” which include but are not limited to: mild to moderateanxiety, anxiety disorder due to a general medical condition, anxietydisorder not otherwise specified, generalized anxiety disorder, panicattack, panic disorder with agoraphobia, panic disorder withoutagoraphobia, posttraumatic stress disorder, social phobia, specificphobia, substance-induced anxiety disorder, acute alcohol withdrawal,obsessive compulsive disorder, agoraphobia, bipolar disorder I or II,bipolar disorder not otherwise specified, cyclothymic disorder,depressive disorder, major depressive disorder, mood disorder,substance-induced mood disorder. In addition, anxiety disorders mayapply to personality disorders including but not limited to thefollowing types: paranoid, antisocial, avoidant behavior, borderlinepersonality disorders, dependent, histronic, narcissistic,obsessive-compulsive, schizoid, and schizotypal.

Procedure:

Behavioral screens were performed on a cohort of 4 wild type, 4heterozygous and 8 homozygous mutant mice. All behavioral tests weredone between 12 and 16 weeks of age unless reduced viabilitynecessitates earlier testing. These tests included open field to measureanxiety, activity levels and exploration.

Prepulse Inhibition of the Acoustic Startle Reflex

Prepulse inhibition of the acoustic startle reflex occurs when a loud120 decibel (dB) startle-inducing tone is preceded by a softer(prepulse) tone. The PPI paradigm consists of six different trial types(70 dB background noise, 120 dB alone, 74 dB+120 dB−pp4, 78 dB+120dB−pp8, 82 dB+120 dB−pp12, and 90 dB+120 dB−pp20) each repeated inpseudo random order six times for a total of 36 trials. The max responseto the stimulus (V max) is averaged for each trial type. Animals with a120 dB average value equal to or below 100 are excluded from analysis.The percent that the prepulse inhibits the animal's response to thestartle stimulus is calculated and graphed.

Results:

PPI: The (−/−) mice exhibited decreased inhibition during pp8, pp12, andpp20 when compared with that of their (+/+) littermates and thehistorical means, suggesting impaired sensorimotor gating/attention inthe mutants.

48.33. Generation and Analysis of Mice Comprising DNA92264-2616(UNQ1932) Gene Disruptions

In these knockout experiments, the gene encoding PRO4407 polypeptides(designated as DNA92264-2616) (UNQ1932) was disrupted. The gene specificinformation for these studies is as follows: the mutated mouse genecorresponds to nucleotide reference: NM_(—)175408 ACCESSION:NM_(—)175408 NID: gi 31341822 ref NM_(—)175408.2 Mus musculus RIKEN cDNAA930027H06 gene (A930027H06Rik); protein reference: Q8C6T0 ACCESSION:Q8C6T0 NID: Mus musculus (Mouse). Hypothetical protein; the human genesequence reference: NM_(—)153345 ACCESSION: NM_(—)153345 NID: gi23503270 refNM_(—)153345.1 Homo sapiens hypothetical protein FLJ90586(FLJ90586); the human protein sequence corresponds to reference: Q81V31ACCESSION: Q8IV31 NID: Homo sapiens (Human). Hypothetical protein.

The mouse gene of interest is RIKEN cDNA A930027H06 gene, ortholog ofhuman hypothetical protein FLJ90586.

Hypothetical protein FLJ90586 contains a weakly predicted signal peptideand an overlapping transmembrane segment. The hypothetical protein maybe secreted or may be located on the plasma membrane (Clark et al,Genome Res 13(10):2265-70 (2003)).

Targeted or gene trap mutations are generated in strain129SvEv^(Brd)-derived embryonic stem (ES) cells. The chimeric mice arebred to C57BL/6J albino mice to generate F1 heterozygous animals. Theseprogeny are intercrossed to generate F2 wild type, heterozygous, andhomozygous mutant progeny. On rare occasions, for example when very fewF1 mice are obtained from the chimera, F1 heterozygous mice are crossedto 129SvEv^(Brd)/C57 hybrid mice to yield additional heterozygousanimals for the intercross to generate the F2 mice. Level I phenotypicanalysis is performed on mice from this generation

wt het hom Total Observed 26 40 13 79 Expected 19.75 39.5 19.75 79Chi-Sq. = 4.21 Significance = 0.12184567 (hom/n) = 0.21 Avg. Litter Size= 9Mutation InformationMutation Type: Homologous Recombination (standard)Description: Coding exons 1 and 2 were targeted (NCBI accessionsBY032793 and NM_(—)175408.2).1. Wild-type Expression Panel: Expression of the target gene wasdetected in all 13 adult tissue samples tested by RT-PCR, except spinalcord, skeletal muscle, and adipose.2. QC Expression: QC Images: Disruption of the target gene was confirmedby Southern hybridization analysis.

48.33.1. Phenotypic Analysis (for Disrupted Gene: DNA92264-2616(UNQ1932)

(a) Overall Phenotypic Summary:

Mutation of the gene encoding the ortholog of human hypothetical proteinFLJ90586 resulted in both heterozygous (+/−) and homozygous (−/−) miceexhibiting increased mean serum glucose levels. Immunologicalabnormalities were also observed in the mutant mice with decreasedpercentages of a subset of B cells in the peritoneal lavage. Genedisruption was confirmed by Southern blot.

(b) Immunology Phenotypic Analysis

Immune related and inflammatory diseases are the manifestation orconsequence of fairly complex, often multiple interconnected biologicalpathways which in normal physiology are critical to respond to insult orinjury, initiate repair from insult or injury, and mount innate andacquired defense against foreign organisms. Disease or pathology occurswhen these normal physiological pathways cause additional insult orinjury either as directly related to the intensity of the response, as aconsequence of abnormal regulation or excessive stimulation, as areaction to self, or as a combination of these.

Though the genesis of these diseases often involves multistep pathwaysand often multiple different biological systems/pathways, interventionat critical points in one or more of these pathways can have anameliorative or therapeutic effect. Therapeutic intervention can occurby either antagonism of a detrimental process/pathway or stimulation ofa beneficial process/pathway.

T lymphocytes (T cells) are an important component of a mammalian immuneresponse. T cells recognize antigens which are associated with aself-molecule encoded by genes within the major histocompatibilitycomplex (MHC). The antigen may be displayed together with MHC moleculeson the surface of antigen presenting cells, virus infected cells, cancercells, grafts, etc. The T cell system eliminates these altered cellswhich pose a health threat to the host mammal. T cells include helper Tcells and cytotoxic T cells. Helper T cells proliferate extensivelyfollowing recognition of an antigen-MHC complex on an antigen presentingcell. Helper T cells also secrete a variety of cytokines, i.e.,lymphokines, which play a central role in the activation of B cells,cytotoxic T cells and a variety of other cells which participate in theimmune response.

In many immune responses, inflammatory cells infiltrate the site ofinjury or infection. The migrating cells may be neutrophilic,eosinophilic, monocytic or lymphocytic as can be determined byhistological examination of the affected tissues. Current Protocols inImmunology, ed. John E. Coligan, 1994, John Wiley & Sons, Inc.

Many immune related diseases are known and have been extensivelystudied. Such diseases include immune-mediated inflammatory diseases(such as rheumatoid arthritis, immune mediated renal disease,hepatobiliary diseases, inflammatory bowel disease (IBD), psoriasis, andasthma), non-immune-mediated inflammatory diseases, infectious diseases,immunodeficiency diseases, neoplasia, and graft rejection, etc. In thearea of immunology, targets were identified for the treatment ofinflammation and inflammatory disorders.

In the area of immunology, targets have been identified herein for thetreatment of inflammation and inflammatory disorders. Immune relateddiseases, in one instance, could be treated by suppressing the immuneresponse. Using neutralizing antibodies that inhibit molecules havingimmune stimulatory activity would be beneficial in the treatment ofimmune-mediated and inflammatory diseases. Molecules which inhibit theimmune response can be utilized (proteins directly or via the use ofantibody agonists) to inhibit the immune response and thus ameliorateimmune related disease.

The following test was performed:

Fluorescence-Activated Cell-Sorting (FACS) Analysis

Procedure:

FACS analysis of immune cell composition from peripheral blood wasperformed including CD4, CD8 and T cell receptor to evaluate Tlymphocytes, CD19 for B lymphocytes, CD45 as a leukocyte marker and panNK for natural killer cells. The FACS analysis was carried out on 2 wildtype and 6 homozygous mice and included cells derived from thymus,spleen, bone marrow and lymph node.

In these studies, analyzed cells were isolated from thymus, peripheralblood, spleen, bone marrow and lymph nodes. Flow cytometry was designedto determine the relative proportions of CD4 and CD8 positive T cells, Bcells, NK cells and monocytes in the mononuclear cell population. ABecton-Dickinson FACS Calibur 3-laser FACS machine was used to assessimmune status. For Phenotypic Assays and Screening, this machine recordsCD4+/CD8−, CD8+/CD4−, NK, B cell and monocyte numbers in addition to theCD4+/CD8+ ratio.

The mononuclear cell profile was derived by staining a single sample oflysed peripheral blood from each mouse with a panel of sixlineage-specific antibodies: CD45 PerCP, anti-TCRb APC, CD4 PE, CD8FITC, pan-NK PE, and CD19 FITC. The two FITC and PE labeled antibodiesstain mutually exclusive cell types. The samples were analyzed using aBecton Dickinson FACS Calibur flow cytometer with CellQuest software.

Results:

Tissue Specific FACS-Project: The (−/−) mice exhibited a decreasedpercentage of B220 Med CD23− cells in peritoneal lavage when comparedwith that of their (+/+) littermates.

These results indicate that the knockout mice exhibited a decrease in asubset of B cells. Thus, the mutant homozygous mice exhibitedimmunological abnormalities associated with decreased levels of B cellprogenitor cells.

These results show that knockout (−/−) mice exhibit immunologicalabnormalities compared to their wild-type (+/+) littermates. Antagonists(inhibitors) of PRO4407 polypeptides would be expected to mimic thisphenotype. PRO4407 polypeptides or agonists thereof appear to act as apositive regulator of B cell development and would be useful in thedevelopment or maturation of B cells which could then participate infast immune responses.

(c) Phenotypic Analysis: Metabolism—Blood Chemistry

In the area of metabolism, targets may be identified for the treatmentof diabetes. Blood chemistry phenotypic analysis includes blood glucosemeasurements. The COBAS Integra 400 (mfr: Roche) was used for runningblood chemistry tests on the mice. In the area of metabolism, targetsmay be identified for the treatment of diabetes.

Results:

Blood Chemistry: The female heterozygous (+/−) and homozygous (−/−) miceexhibited an increased mean serum glucose level (˜2 SD above littermatewild-type mice) when compared with that of their gender-matched (+/+)littermates and the historical mean. Thus, both the heterozygous andhomozygous (−/−) mice showed a negative phenotype related to abnormalglucose metabolism. However, female serum insulin and urine glucoselevels were normal.48.34. Generation and Analysis of Mice Comprising DNA93011-2637(UNQ1942) Gene Disruptions

In these knockout experiments, the gene encoding PRO4425 polypeptides(designated as DNA93011-2637) (UNQ1942) was disrupted. The gene specificinformation for these studies is as follows: the mutated mouse genecorresponds to nucleotide reference: XM_(—)132070 ACCESSION:XM_(—)132070NID: gi 51710957 ref XM_(—)132070.3 PREDICTED: Mus musculus RIKEN cDNA4930443F05 gene (4930443F05Rik); protein reference: XP_(—)132070 similarto Cytokine-like protein C17 precursor (UNQ1942/PRO4425) [Mus musculus];the human gene sequence reference: NM_(—)018659 Homo sapienscytokine-like protein C17 (C17); the human protein sequence correspondsto reference: Q9NRR1 ACCESSION: Q9NRR1 NID: Homo sapiens (Human).Cytokine-like protein C17 precursor (UNQ1942/PRO4425)

The mouse gene of interest is RIKEN cDNA 4930443F05 gene, ortholog ofhuman C17 (cytokine-like protein C17). Aliases include Gm147.

C17 is a protein secreted by CD34 mononuclear stem cells that likelyfunctions as a signal-transducing ligand. The protein contains a signalpeptide and generally shares structural similarities with othercytokines. Expression of C17 is up-regulated by cytokines that maintainstem cells and is down-regulated by hematopoietic colony-stimulatingfactors that stimulate differentiation of stem cells (Liu et al,Genomics 65(3):283-92 (2000)). C17 may play a role in hematopoiesis orimmunity.

Targeted or gene trap mutations are generated in strain129SvEv^(Brd)-derived embryonic stem (ES) cells. The chimeric mice arebred to C57BL/6J albino mice to generate F1 heterozygous animals. Theseprogeny are intercrossed to generate F2 wild type, heterozygous, andhomozygous mutant progeny. On rare occasions, for example when very fewF1 mice are obtained from the chimera, F1 heterozygous mice are crossedto 129SvEv^(Brd)/C57 hybrid mice to yield additional heterozygousanimals for the intercross to generate the F2 mice. Level I phenotypicanalysis is performed on mice from this generation

wt het hom Total Observed 19 47 22 88 Expected 22 44 22 88 Chi-Sq. =3.43 Significance = 0.17996371 (hom/n) = 0.23 Avg. Litter Size = 9Mutation InformationMutation Type: Homologous Recombination (standard)Description: Coding exons 2 through 4 were targeted (NCBI accessionBC063103.1).1. Wild-type Expression Panel: Expression of the target gene wasdetected in embryonic stem (ES) cells and in all 13 adult tissue samplestested by RT-PCR, except skeletal muscle.2. QC Expression: QC Images: Disruption of the target gene was confirmedby Southern hybridization analysis.

48.34.1. Phenotypic Analysis (for Disrupted Gene: DNA93011-2637(UNQ1942)

(a) Overall Phenotypic Summary:

Mutation of the gene encoding the ortholog of human cytokine-likeprotein C17 (C17) resulted in an increased percentage of CD4 cells inthe peripheral blood, increased TCRbeta+ in the thymus, increasedCD11b+CD11c+ and increased natural killer cells in the lymph nodes, andincreased percentage of CD117+ cells in the peritoneal lavage in the(−/−) mice. Decreased IgG2a and increased IgA mean serum levels werealso shown in the (−/−) mice. The (−/−) mice also exhibited an increasedretinal artery-to-vein ratio. The mutant (−/−) mice also exhibitedincreased trabecular number and connectivity density. Gene disruptionwas confirmed by Southern blot.

(b) Expression

C17 is expressed in arterial endothelium as shown by ISH studies. TheChr4 neighborhood is rich in genes implicated in bone development. [SeeEXAMPLE 57 for protocol]

(c) Immunology Phenotypic Analysis

Immune related and inflammatory diseases are the manifestation orconsequence of fairly complex, often multiple interconnected biologicalpathways which in normal physiology are critical to respond to insult orinjury, initiate repair from insult or injury, and mount innate andacquired defense against foreign organisms. Disease or pathology occurswhen these normal physiological pathways cause additional insult orinjury either as directly related to the intensity of the response, as aconsequence of abnormal regulation or excessive stimulation, as areaction to self, or as a combination of these.

Though the genesis of these diseases often involves multistep pathwaysand often multiple different biological systems/pathways, interventionat critical points in one or more of these pathways can have anameliorative or therapeutic effect. Therapeutic intervention can occurby either antagonism of a detrimental process/pathway or stimulation ofa beneficial process/pathway.

T lymphocytes (T cells) are an important component of a mammalian immuneresponse. T cells recognize antigens which are associated with aself-molecule encoded by genes within the major histocompatibilitycomplex (MHC). The antigen may be displayed together with MHC moleculeson the surface of antigen presenting cells, virus infected cells, cancercells, grafts, etc. The T cell system eliminates these altered cellswhich pose a health threat to the host mammal. T cells include helper Tcells and cytotoxic T cells. Helper T cells proliferate extensivelyfollowing recognition of an antigen-MHC complex on an antigen presentingcell. Helper T cells also secrete a variety of cytokines, i.e.,lymphokines, which play a central role in the activation of B cells,cytotoxic T cells and a variety of other cells which participate in theimmune response.

In many immune responses, inflammatory cells infiltrate the site ofinjury or infection. The migrating cells may be neutrophilic,eosinophilic, monocytic or lymphocytic as can be determined byhistological examination of the affected tissues. Current Protocols inImmunology, ed. John E. Coligan, 1994, John Wiley & Sons, Inc.

Many immune related diseases are known and have been extensivelystudied. Such diseases include immune-mediated inflammatory diseases(such as rheumatoid arthritis, immune mediated renal disease,hepatobiliary diseases, inflammatory bowel disease (IBD), psoriasis, andasthma), non-immune-mediated inflammatory diseases, infectious diseases,immunodeficiency diseases, neoplasia, and graft rejection, etc. In thearea of immunology, targets were identified for the treatment ofinflammation and inflammatory disorders.

In the area of immunology, targets have been identified herein for thetreatment of inflammation and inflammatory disorders. Immune relateddiseases, in one instance, could be treated by suppressing the immuneresponse. Using neutralizing antibodies that inhibit molecules havingimmune stimulatory activity would be beneficial in the treatment ofimmune-mediated and inflammatory diseases. Molecules which inhibit theimmune response can be utilized (proteins directly or via the use ofantibody agonists) to inhibit the immune response and thus ameliorateimmune related disease.

The following tests were performed:

Fluorescence-Activated Cell-Sorting (FACS) Analysis

Procedure:

FACS analysis of immune cell composition from peripheral blood wasperformed including CD4, CD8 and T cell receptor to evaluate Tlymphocytes, CD19 for B lymphocytes, CD45 as a leukocyte marker and panNK for natural killer cells. The FACS analysis was carried out on 2 wildtype and 6 homozygous mice and included cells derived from thymus,spleen, bone marrow and lymph node.

In these studies, analyzed cells were isolated from thymus, peripheralblood, spleen, bone marrow and lymph nodes. Flow cytometry was designedto determine the relative proportions of CD4 and CD8 positive T cells, Bcells, NK cells and monocytes in the mononuclear cell population. ABecton-Dickinson FACSCalibur 3-laser FACS machine was used to assessimmune status. For Phenotypic Assays and Screening, this machine recordsCD4+/CD8−, CD8+/CD4−, NK, B cell and monocyte numbers in addition to theCD4+/CD8+ ratio.

The mononuclear cell profile was derived by staining a single sample oflysed peripheral blood from each mouse with a panel of sixlineage-specific antibodies: CD45 PerCP, anti-TCRb APC, CD4 PE, CD8FITC, pan-NK PE, and CD19 FITC. The two FITC and PE labeled antibodiesstain mutually exclusive cell types. The samples were analyzed using aBecton Dickinson FACSCalibur flow cytometer with CellQuest software.

Results:

FACS3: The (−/−) mice exhibited an altered distribution of leukocytesubsets in the peripheral blood, characterized by an increased meanpercentage of CD4 cells when compared with that of their (+/+)littermates and the historical mean.

Tissue Specific FACS-Project: The (−/−) mice exhibited an increasedpercentage of TcRbeta+ cells in thymus, increased percentages ofCD11b+CD11c+ and NK cells in lymph node, and an increased percentage ofCD117+ cells in peritoneal lavage when compared with those of their(+/+) littermates.

Thus, knocking out the gene which encodes PRO4425 polypeptides causes anincrease in the T cell population. From these observations, PRO4425polypeptides or the gene encoding PRO4425 appears to act as a negativeregulator of T cell proliferation. Thus, PRO4425 polypeptides oragonists thereof would be beneficial as a negative regulator of T cellproliferation in those instances wherein a pronounced T-cellproliferation is present such as occurs in autoimmune diseases (forexample rheumatoid arthritis patients). In addition, PRO4425polypeptides would be especially useful in preventing skin graftrejections.

In addition, the FACS results indicate that the homozygous mutant micehave a increased mean percentage of natural killer cells. Natural killercells are the first line of defense to viral infection since these cellshave been implicated in viral immunity and in defense against tumors.Natural killer cells or NK cells act as effectors in antibody-dependentcell-mediated cytotoxicity and have been identified by their ability tokill certain lymphoid tumor cell lines in vitro without the need forprior immunization or activation. Thus, PRO4425 polypeptides act as anegative regulator for NK production.

Serum Immunoglobulin Isotyping Assay:

The Serum Immunoglobulin Isotyping Assay is performed using a CytometricBead Array (CBA) kit. This assay is used to rapidly identify the heavyand light chain isotypes of a mouse monoclonal antibody in a singlesample. The values expressed are “relative fluorescence units” and arebased on the detection of kappa light chains. Any value <6 is notsignificant.

Results:

Serum Imm. 2: The (−/−) mice exhibited a decreased mean serum IgG2alevel and a slightly increased mean serum IgA level when compared withthat of their (+/+) littermates, the (+/+) mice within the project run,and the historical medians.

The serum immunoglobulin isotyping assay revealed that homozygous adultsexhibited decreased serum IgG2a levels. Thus, homozygotes showed anabnormally low serum immunoglobulins compared with the (+/+)littermates. Thus, the gene encoding PRO4425 is essential for makingimmunoglobulins (or gamma globulins). Likewise, IgG2a immunoglobulinshave neutralization effects and to a lesser extent are important foractivation of the complement system.

(d) Cardiovascular Phenotypic Analysis:

In the area of cardiovascular biology, phenotypic testing was performedto identify potential targets for the treatment of cardiovascular,endothelial or angiogenic disorders. One such phenotypic test includedoptic fundus photography and angiography to determine the retinalarteriovenous ratio (A/V ratio) in order to flag various eyeabnormalities. An abnormal A/V ratio signals such systemic diseases ordisorders that may be related to the vascular disease of hypertension(and any disease that causes hypertension, e.g. atherosclerosis),diabetes or other ocular diseases corresponding to opthalmologicaldisorders. Such eye abnormalities may include but are not limited to thefollowing: retinal abnormality is retinal dysplasia, variousretinopathies, restenosis, retinal artery obstruction or occlusion;retinal degeneration causing secondary atrophy of the retinalvasculature, retinitis pigmentosa, macular dystrophies, Stargardt'sdisease, congenital stationary night blindness, choroideremia, gyrateatrophy, Leber's congenital amaurosis, retinoschisis disorders, Wagner'ssyndrome, Usher syndromes, Zellweger syndrome, Saldino-Mainzer syndrome,Senior-Loken syndrome, Bardet-Biedl syndrome, Alport's syndrome,Alstom's syndrome, Cockayne's syndrome, dysplaisa spondyloepiphysariacongentia, Flynn-Aird syndrome, Friedreich ataxia, Hallgren syndrome,Marshall syndrome, Albers-Schnoberg disease, Refsum's disease,Kearns-Sayre syndrome, Waardenburg's syndrome, Alagile syndrome,myotonic dystrophy, olivopontocerebellar atrophy, Pierre-Mariedunsdrome, Stickler syndrome, carotinemeia, cystinosis, Wolframsyndrome, Bassen-Kornzweig syndrome, abetalipoproteinemia, incontinentiapigmenti, Batten's disease, mucopolysaccharidoses, homocystinuria, ormannosidosis.

Procedure: A cohort of 4 wild type, 4 heterozygous and 8 homozygous micewere tested in this assay. Optic fundus photography was performed onconscious animals using a Kowa Genesis small animal fundus cameramodified according to Hawes and coauthors (Hawes et al., 1999 MolecularVision 1999; 5:22). Intra-peritoneal injection of fluorescein permittedthe acquisition of direct light fundus images and fluorescent angiogramsfor each examination. In addition to direct opthalmological changes,this test can detect retinal changes associated with systemic diseasessuch as diabetes and atherosclerosis or other retinal abnormalities.Pictures were provided of the optic fundus under normal light. Theangiographic pictures allowed examination of the arteries and veins ofthe eye. In addition an artery to vein (A/V) ratio was determined forthe eye.

Opthalmology analysis was performed on generated F2 wild type,heterozygous, and homozygous mutant progeny using the protocol describedabove. Specifically, the A/V ratio was measured and calculated accordingto the fundus images with Kowa COMIT+ software. This test takes colorphotographs through a dilated pupil: the images help in detecting andclassifying many diseases. The artery to vein ratio (A/V) is the ratioof the artery diameter to the vein diameter (measured before thebifurcation of the vessels). Many diseases will influence the ratio,i.e., diabetes, cardiovascular disorders, papilledema, optic atrophy orother eye abnormalities such as retinal degeneration (known as retinitispigmentosa) or retinal dysplasia, vision problems or blindness. Thus,phenotypic observations which result in an increased artery-to-veinratio in homozygous (−/−) and heterozygous (+/−) mutant progeny comparedto wild-type (+/+) littermates would be indicative of such pathologicalconditions.

Results:

Fundus: The (−/−) mice exhibited an increased mean retinalartery-to-vein ratio when compared with that of their (+/+) littermatesand the historical mean.

In this study, the (−/−) exhibited an increased mean artery-to-vein(A/V) ratio when compared with their (+/+) littermates indicatingretinal degeneration. In summary, by knocking out the gene identified asDNA93011-2637 encoding PRO4425 polypeptides, homozygous mutant progenyexhibit phenotypes which are associated with retinal degeneration. Suchdetected retinal changes are most commonly associated withcardiovascular systemic diseases or disorders that may be related to thevascular disease of hypertension (and any disease that causeshypertension, e.g. atherosclerosis), diabetes or other ocular diseasescorresponding to opthalmological disorders such as retinal degeneration.Thus, antagonists (inhibitors) of PRO4425 encoding genes would lead tosimilar pathological retinal changes, whereas agonists may be useful astherapeutic agents in the treatment of hypertension, atherosclerosis orother opthalmological disorders including retinal degeneration anddiseases associated with this condition (as indicated above).

(e) Bone Metabolism: Radiology Phenotypic Analysis

In the area of bone metabolism, targets were identified herein for thetreatment of arthritis, osteoporosis, osteopenia and osteopetrosis aswell as identifying targets that promote bone healing. Tests included:

DEXA for measurement of bone mineral density on femur and vertebra

MicroCT for very high resolution and very high sensitivity measurementsof bone mineral density for both trabecular and cortical bone.

Dexa Analysis—Test Description:

Procedure: A cohort of 4 wild type, 4 heterozygous and 8 homozygous micewere tested in this assay. Dual Energy X-ray Absorptiometry (DEXA) hasbeen used successfully to identify changes in bone. Anesthetized animalswere examined and bone mineral content (BMC), BMC/LBM ratios, volumetricbone mineral density (vBMD), total body BMD, femur BMD and vertebra BMDwere measured.

The mouse was anesthetized by intraperitoneal injection of Avertin(1.25% 2,2,2,-tribromoethanol, 20 ml/kg body weight), body length andweight were measured, and then the mouse was placed in a prone positionon the platform of the PIXImus™ Densitometer (Lunar Inc.) for a DEXAscan. Using Lunar PIXImus software, the bone mineral density (BMD) andfat composition (% fat) and total tissue mass (TTM) were determined inthe regions of interest (ROI) [i.e., whole body, vertebrae, and bothfemurs].

Bone MicroCT Analysis:

Procedure: MicroCT was also used to get very sensitive measurements ofBMD. One vertebra and 1 femur were taken from a cohort of 4 wild typeand 8 homozygous mice. Measurements were taken of lumbar 5 vertebratrabecular bone volume, trabecular thickness, connectivity density andmidshaft femur total bone area and cortical thickness. The μCT40 scansprovided detailed information on bone mass and architecture. Multiplebones were placed into sample holders and scanned automatically.Instrument software was used to select regions of interest for analysis.Trabecular bone parameters were analyzed in the fifth lumbar vertebrae(LV5) at 16 micrometer resolution and cortical bone parameters wereanalyzed in the femur midshaft at a resolution of 20 micrometers.

Results:

Micro CT: Male (−/−) mice exhibited increased trabecular number andconnectivity density.

In summary, the (−/−) mice exhibited increased trabecular bone mineraldensity when compared with their gender-matched (+/+) littermates. Theseresults indicate that the knockout mutant phenotype may be associatedwith such bone abnormalities as osteopetrosis. Osteopetrosis is acondition characterized by abnormal thickening and hardening of bone andabnormal fragility of the bones. As such, PRO4425 polypeptides oragonists thereof would be beneficial for the treatment of osteopetrosisor other osteo-related diseases. On the other hand, inhibitors orantagonists of PRO4425 polypeptides would be useful in bone healing.

48.35. Generation and Analysis of Mice Comprising DNA59770-2652(UNQ2426) Gene Disruptions

In these knockout experiments, the gene encoding PRO4985 polypeptides(designated as DNA59770-2652) (UNQ2426) was disrupted. The gene specificinformation for these studies is as follows: the mutated mouse genecorresponds to nucleotide reference: XM_(—)203978 PREDICTED: Musmusculus RIKEN cDNA 5330417C22 gene (5330417C22Rik); protein reference:XP_(—)203978 RIKEN cDNA 5330417C22 gene [Mus musculus]; the human genesequence reference: NM_(—)020775 Homo sapiens maba1 (KIAA1324); thehuman protein sequence corresponds to reference: NP_(—)065826 maba1[Homo sapiens].

The mouse gene of interest is RIKEN cDNA 5330417C22 gene, ortholog ofhuman maba1. Aliases include KIAA1324 and RP11-352P4.1.

Maba1 is a putative integral plasma membrane protein, consisting of asignal peptide, a large extracellular region containing a keratin/highsulfur B2 protein (B2) domain, a transmembrane segment, and a shortcytoplasmic C-terminus. Proteins with B2 domains include thekeratin/high sulfur B2 family of proteins, which function as componentsof hair fibers synthesized by differentiating hair cells (Mitsui et al,Gene 208(2): 123-9 (1998); Rogers et al, J Biol Chem 276(22):19440-51(2001); Shibuya et al, Genomics 83(4):679-93 (2004)).

Targeted or gene trap mutations are generated in strain129SvEv^(Brd)-derived embryonic stem (ES) cells. The chimeric mice arebred to C57BL/6J albino mice to generate F1 heterozygous animals. Theseprogeny are intercrossed to generate F2 wild type, heterozygous, andhomozygous mutant progeny. On rare occasions, for example when very fewF1 mice are obtained from the chimera, F1 heterozygous mice are crossedto 129SvEv^(Brd)/C57 hybrid mice to yield additional heterozygousanimals for the intercross to generate the F2 mice. Level I phenotypicanalysis is performed on mice from this generation

wt het hom Total Observed 18 43 30 91 Expected 22.75 45.5 22.75 91Chi-Sq. = 0.52 Significance = 0.7710516 (hom/n) = 0.26 Avg. Litter Size= 9Mutation InformationMutation Type: Homologous Recombination (standard)Description: Coding exon 1 was targeted (NCBI accession XM_(—)203978.4).1. Wild-type Expression Panel: Expression of the target gene wasdetected in all 26 adult tissue samples tested by RT-PCR, exceptskeletal muscle, bone, heart, adipose, blood, banded heart, aortic tree,MG 5 week virgin, MG mature virgin, MG 3 day post-partum (lactating), MG3 day post-weaning (early involution), and MG 7 day post-weaning (lateinvolution).2. QC Expression: QC Images: Disruption of the target gene was confirmedby Southern hybridization analysis.

48.35.1. Phenotypic Analysis (for Disrupted Gene: DNA59770-2652(UNQ2426)

(a) Overall Phenotypic Summary:

Mutation of the gene encoding the ortholog of human maba1 resulted indefective spermatogenesis in male (−/−) mice. Microscopic analysisrevealed defective spermatogenesis in the testis and hypospermia anddefective spermatozoa in the epididymus of the homozygous mutant mice,consistent with the infertility noted in the male homozygous mutantclinically. The (−/−) mice also exhibited decreased body fat. Disruptionof the target gene was confirmed by Southern hybridization analysis.

(b) Pathology

Microscopic: The male (−/−) mice exhibited moderate diffuse defectivespermatogenesis in the testes and hypospermia and defective spermatozoain the epididymus. The male (−/−) mice exhibited a late-stage defect inspermatogenesis resulting in failure to develop beyond the roundspermatid stage. The spermatocytes exhibited large rounded heads andlarge cytoplasmic droplets.Gene Expression: LacZ activity was not detected in the panel of tissuesby immunohistochemical analysis.

(c) Body Diagnostics

Fertility: The male (−/−) mouse produced no pups after 60 days ofbreeding and 4 matings with female (+/+) mice. The mouse appearedhealthy, and a penile erection could be induced by abdominal pressure.

Bone Metabolism: Radiology Phenotypic Analysis

In the area of bone metabolism, targets were identified herein for thetreatment of arthritis, osteoporosis, osteopenia and osteopetrosis aswell as identifying targets that promote bone healing. Tests included:

DEXA for measurement of bone mineral density on femur and vertebra

MicroCT for very high resolution and very high sensitivity measurementsof bone mineral density for both trabecular and cortical bone.

Dexa Analysis—Test Description:

Procedure: A cohort of 4 wild type, 4 heterozygous and 8 homozygous micewere tested in this assay. Dual Energy X-ray Absorptiometry (DEXA) hasbeen used successfully to identify changes in bone. Anesthetized animalswere examined and bone mineral content (BMC), BMC/LBM ratios, volumetricbone mineral density (vBMD), total body BMD, femur BMD and vertebra BMDwere measured.

The mouse was anesthetized by intraperitoneal injection of Avertin(1.25% 2,2,2,-tribromoethanol, 20 ml/kg body weight), body length andweight were measured, and then the mouse was placed in a prone positionon the platform of the PIXImus™ Densitometer (Lunar Inc.) for a DEXAscan. Using Lunar PIXImus software, the bone mineral density (BMD) andfat composition (% fat) and total tissue mass (TTM) were determined inthe regions of interest (ROI) [i.e., whole body, vertebrae, and bothfemurs].

Results:

DEXA: The male (−/−) mice exhibited decreased mean percent total bodyfat and total fat mass when compared with the levels for theirgender-matched (+/+) littermates and the historical mean.

Mutant (−/−) mice deficient in the gene encoding PRO4985 polypeptidesshow a phenotype consistent with tissue wasting diseases (decreasedtotal body fat (%) and fat mass (g)) or abnormal lipid metabolism. Thus,antagonists or inhibitors of PRO4985 polypeptides or its encoding genewould mimic these metabolic and developmental related effects. On theother hand, PRO4985 polypeptides or agonists thereof would be useful inthe prevention and/or treatment of such metabolic disorders and fornormal male reproductive development.

48.36. Generation and Analysis of Mice Comprising DNA80135-2655(UNQ2429) Gene Disruptions

In these knockout experiments, the gene encoding PRO4989 polypeptides(designated as DNA80135-2655) (UNQ2429) was disrupted. The gene specificinformation for these studies is as follows: the mutated mouse genecorresponds to nucleotide reference: NM_(—)153542 ACCESSION:NM_(—)153542 NID: gi 23956307 ref NM_(—)153542.1 Mus musculushypothetical protein MGC25719 (MGC25719); protein reference: Q8CI70ACCESSION: Q8CI70 NID: Mus musculus (Mouse). Leucine rich repeatcontaining 20; the human gene sequence reference: NM_(—)018205ACCESSION: NM_(—)018205 NID: gi 8922643 ref NM_(—)018205.1 Homo sapienshypothetical protein FLJ10751 (FLJ10751); the human protein sequencecorresponds to reference: Q8TCA0 ACCESSION: Q8TCA0 NID: Homo sapiens(Human). Hypothetical protein FLJ37415.

The mouse gene of interest is Lrrc20 (leucine rich repeat containing20), ortholog of human LRRC20. Aliases include MGC25719, cDNA sequenceBC036304, FLJ10751, and FLJ10844.

LRRC20 is a putative extracellular protein of 184 amino acids,containing leucine-rich repeats (Clark et al, Genome Res 13(10):2265-70(2003)).

Targeted or gene trap mutations are generated in strain129SvEv^(Brd)-derived embryonic stem (ES) cells. The chimeric mice arebred to C57BL/6J albino mice to generate F1 heterozygous animals. Theseprogeny are intercrossed to generate F2 wild type, heterozygous, andhomozygous mutant progeny. On rare occasions, for example when very fewF1 mice are obtained from the chimera, F1 heterozygous mice are crossedto 129SvEv^(Brd)/C57 hybrid mice to yield additional heterozygousanimals for the intercross to generate the F2 mice. Level I phenotypicanalysis is performed on mice from this generation

wt het hom Total Observed 21 40 23 84 Expected 21 42 21 84 Chi-Sq. =1.86 Significance = 0.39455372 (hom/n) = 0.26 Avg. Litter Size = 10Mutation InformationMutation Type Homologous Recombination (standard)Description: Coding exon 1 was targeted (NCBI accession NM_(—)153542.1).1. Wild-type Expression Panel: Expression of the target gene wasdetected in all 13 adult tissue samples tested by RT-PCR, except boneand adipose.2. QC Expression: QC Images: Disruption of the target gene was confirmedby Southern hybridization analysis.

48.36.1. Phenotypic Analysis (for Disrupted Gene: DNA80135-2655(UNQ2429)

(a) Overall Phenotypic Summary:

Mutation of the gene encoding the ortholog of human leucine rich repeatcontaining 20 (LRRC20) resulted in the (−/−) mice exhibited decreasedanxiety during open filed testing. Gene disruption was confirmed bySouthern blot.

(b) Phenotypic Analysis: CNS/Neurology

In the area of neurology, analysis focused herein on identifying in vivovalidated targets for the treatment of neurological and psychiatricdisorders including depression, generalized anxiety disorders, attentiondeficit hyperactivity disorder, obsessive compulsive disorder,schizophrenia, cognitive disorders, hyperalgesia and sensory disorders.Neurological disorders include the category defined as “anxietydisorders” which include but are not limited to: mild to moderateanxiety, anxiety disorder due to a general medical condition, anxietydisorder not otherwise specified, generalized anxiety disorder, panicattack, panic disorder with agoraphobia, panic disorder withoutagoraphobia, posttraumatic stress disorder, social phobia, specificphobia, substance-induced anxiety disorder, acute alcohol withdrawal,obsessive compulsive disorder, agoraphobia, bipolar disorder I or II,bipolar disorder not otherwise specified, cyclothymic disorder,depressive disorder, major depressive disorder, mood disorder,substance-induced mood disorder. In addition, anxiety disorders mayapply to personality disorders including but not limited to thefollowing types: paranoid, antisocial, avoidant behavior, borderlinepersonality disorders, dependent, histronic, narcissistic,obsessive-compulsive, schizoid, and schizotypal.

Procedure:

Behavioral screens were performed on a cohort of 4 wild type, 4heterozygous and 8 homozygous mutant mice. All behavioral tests weredone between 12 and 16 weeks of age unless reduced viabilitynecessitates earlier testing. These tests included open field to measureanxiety, activity levels and exploration.

Open Field Test:

Several targets of known drugs have exhibited phenotypes in the openfield test. These include knockouts of the seratonin transporter, thedopamine transporter (Giros et al., Nature. 1996 Feb. 15;379(6566):606-12), and the GABA receptor (Homanics et al., Proc NatlAcad Sci USA. 1997 Apr. 15; 94(8):4143-8). An automated open-field assaywas customized to address changes related to affective state andexploratory patterns related to learning. First, the field (40×40 cm)was selected to be relatively large for a mouse, thus designed to pickup changes in locomotor activity associated with exploration. Inaddition, there were 4 holes in the floor to allow for nose-poking, anactivity specifically related to exploration. Several factors were alsodesigned to heighten the affective state associated with this test. Theopen-field test is the first experimental procedure in which the miceare tested, and the measurements that were taken were the subjects'first experience with the chamber. In addition, the open-field wasbrightly lit. All these factors will heighten the natural anxietyassociated with novel and open spaces. The pattern and extent ofexploratory activity, and especially the center-to-total distancetraveled ratio, may then be able to discern changes related tosusceptibility to anxiety or depression. A large arena (40 cm×40 cm,VersaMax animal activity monitoring system from AccuScan Instruments)with infrared beams at three different levels was used to recordrearing, hole poke, and locomotor activity. The animal was placed in thecenter and its activity was measured for 20 minutes. Data from this testwas analyzed in five, 4-minute intervals. The total distance traveled(cm), vertical movement number (rearing), number of hole pokes, and thecenter to total distance ratio were recorded.

The propensity for mice to exhibit normal habituation responses to anovel environment is assessed by determining the overall change in theirhorizontal locomotor activity across the 5 time intervals. Thiscalculated slope of the change in activity over time is determined usingnormalized, rather than absolute, total distance traveled. The slope isdetermined from the regression line through the normalized activity ateach of the 5 time intervals. Normal habituation is represented by anegative slope value.

Results:

The (−/−) mice exhibited an increased median sum time and distancein-center during open field testing when compared with theirgender-matched (+/+) littermates and the historical mean, suggesting adecreased anxiety-like response in the mutants.

The (−/−) mice exhibited an increased median sum time in the center areawhen compared with their gender-matched (+/+) littermates, which isindicative of a decreased anxiety-like response in the mutants. Thus,knockout mice demonstrated a phenotype consistent with depression,generalized anxiety disorders, cognitive disorders, hyperalgesia andsensory disorders and/or bipolar disorders. Thus, PRO4989 polypeptidesand agonists thereof would be useful for the treatment or ameliorationof the symptoms associated with depressive disorders.

48.37. Generation and Analysis of Mice Comprising DNA92929-2534-1(UNQ2456) Gene Disruptions

In these knockout experiments, the gene encoding PRO5737 polypeptides(designated as DNA92929-2534-1) (UNQ2456) was disrupted. The genespecific information for these studies is as follows: the mutated mousegene corresponds to nucleotide reference: NM_(—)153511 ACCESSION:NM_(—)153511 NID: gi 23943829 ref NM_(—)153511.1 Mus musculusinterleukin 1 family, member 9 (IL1F9); protein reference: Q8R460ACCESSION: Q8R460NID: Mus musculus (Mouse). Interleukin 1 family member9 (IL-1F9); the human gene sequence reference: NM_(—)019618 Homo sapiensinterleukin 1 family, member 9 (IL1F9); the human protein sequencecorresponds to reference: Q9NZH8 ACCESSION: Q9NZH8 NID: Homo sapiens(Human). Interleukin 1 family member 9 (IL-1F9) (Interleukin-1homolog 1) (IL-1H1) (Interleukin-1 epsilon) (IL-1 epsilon) (IL-1 relatedprotein 2) (IL-1RP2).

The mouse gene of interest is Il1f9 (interleukin 1 family, member 9),ortholog of human IL1F9. Aliases include IL-1F9, IL1E, IL1H1, IL-1H1,IL1RP2, IL-1RP2, IL-1-epsilon, IL-1 (EPSILON), interleukin-1 epsilon,IL-1 related protein 2, interleukin-1 homolog 1, and interleukin1-related protein 2.

IL1F9 is a secreted protein that functions as a ligand for receptorIL1RL2, activating nuclear factor kappaB. IL1F9 is expressed inepithelia from a variety of tissues, such as skin, lung, stomach andesophagus, and is induced by tumor necrosis factor-alpha and byinterferon-gamma in keratinocytes. IL1F9 likely plays a role in immunefunction and inflammation (Debets et al, J Immunol 167(3):1440-6 (2001);Kumar et al, J Biol Chem 275(14):10308-14 (2000); Towne et al, J BiolChem 279(14):13677-88 (2004)).

Targeted or gene trap mutations are generated in strain129SvEv^(Brd)-derived embryonic stem (ES) cells. The chimeric mice arebred to C57BL/6J albino mice to generate F1 heterozygous animals. Theseprogeny are intercrossed to generate F2 wild type, heterozygous, andhomozygous mutant progeny. On rare occasions, for example when very fewF1 mice are obtained from the chimera, F1 heterozygous mice are crossedto 129SvEv^(Brd)/C57 hybrid mice to yield additional heterozygousanimals for the intercross to generate the F2 mice. Level I phenotypicanalysis is performed on mice from this generation

wt het hom Total Observed 17 39 21 77 Expected 19.25 38.5 19.25 77Chi-Sq. = 1.41 Significance = 0.4941086 (hom/n) = 0.28 Avg. Litter Size= 9Mutation InformationMutation Type: Homologous Recombination (standard)Description: Coding exons 1 through 3 were targeted (NCBI accessionNM_(—)153511.1).1. Wild-type Expression Panel: Expression of the target gene wasdetected in all 13 adult tissue samples tested by RT-PCR, exceptskeletal muscle and adipose.2. QC Expression: QC Images: Disruption of the target gene was confirmedby Southern hybridization analysis.

48.37.1. Phenotypic Analysis (for Disrupted Gene: DNA92929-2534-1(UNQ2456)

(a) Overall Phenotypic Summary:

Mutation of the gene encoding the ortholog of human interleukin 1family, member 9 (IL1F9) resulted in the mutant (−/−) mice exhibitingdecreased lean body mass. Gene disruption was confirmed by Southernblot.

(b) Expression

UNQ2456 is upregulated in squamous cell carcinoma or dysplasia (in headand neck); squamous cell carcinoma (in lung); and hyperplasia of adenoidtonsils. UNQ2456 is also upregulated in psoriasis. [See EXAMPLES 54 and55 for protocol]

(c) Bone Metabolism & Body Diagnostics: Radiology Phenotypic Analysis

In the area of bone metabolism, targets were identified herein for thetreatment of arthritis, osteoporosis, osteopenia and osteopetrosis aswell as identifying targets that promote bone healing. Tests included:

DEXA for measurement of bone mineral density on femur and vertebra

MicroCT for very high resolution and very high sensitivity measurementsof bone mineral density for both trabecular and cortical bone.

Dexa Analysis—Test Description:

Procedure: A cohort of 4 wild type, 4 heterozygous and 8 homozygous micewere tested in this assay. Dual Energy X-ray Absorptiometry (DEXA) hasbeen used successfully to identify changes in bone. Anesthetized animalswere examined and bone mineral content (BMC), BMC/LBM ratios, volumetricbone mineral density (vBMD), total body BMD, femur BMD and vertebra BMDwere measured.

The mouse was anesthetized by intraperitoneal injection of Avertin(1.25% 2,2,2,-tribromoethanol, 20 ml/kg body weight), body length andweight were measured, and then the mouse was placed in a prone positionon the platform of the PIXImus™ Densitometer (Lunar Inc.) for a DEXAscan. Using Lunar PIXImus software, the bone mineral density (BMD) andfat composition (% fat) and total tissue mass (TTM) were determined inthe regions of interest (ROI) [i.e., whole body, vertebrae, and bothfemurs].

Bone MicroCT Analysis:

Procedure: MicroCT was also used to get very sensitive measurements ofBMD. One vertebra and 1 femur were taken from a cohort of 4 wild typeand 8 homozygous mice. Measurements were taken of lumbar 5 vertebratrabecular bone volume, trabecular thickness, connectivity density andmidshaft femur total bone area and cortical thickness. The μCT40 scansprovided detailed information on bone mass and architecture. Multiplebones were placed into sample holders and scanned automatically.Instrument software was used to select regions of interest for analysis.Trabecular bone parameters were analyzed in the fifth lumbar vertebrae(LV5) at 16 micrometer resolution and cortical bone parameters wereanalyzed in the femur midshaft at a resolution of 20 micrometers.

Results:

DEXA: The male (−/−) mice exhibited decreased mean lean body mass whencompared with that of their gender-matched (+/+) littermates and thehistorical mean.

Mutant (−/−) mice deficient in the gene encoding PRO5737 polypeptidesshow a phenotype consistent with tissue wasting diseases (decreased leanbody mass). Thus, antagonists or inhibitors of PRO5737 polypeptides orits encoding gene would mimic these metabolic related effects. On theother hand, PRO5737 polypeptides or agonists thereof would be useful inthe prevention and/or treatment of such metabolic disorders as cachexiaor other tissue wasting diseases.

48.38. Generation and Analysis of Mice Comprising DNA108912-2680(UNQ2500) Gene Disruptions

In these knockout experiments, the gene encoding PRO5800 polypeptides(designated as DNA108912-2680) (UNQ2500) was disrupted. The genespecific information for these studies is as follows: the mutated mousegene corresponds to nucleotide reference: NM_(—)023304 ACCESSION:NM_(—)023304 NID: gi 12963626 ref NM_(—)023304.1 Mus musculus fibroblastgrowth factor 22 (Fgf22); protein reference: Q9ESS2 ACCESSION: Q9ESS2NID: Mus musculus (Mouse). FIBROBLAST GROWTH FACTOR-22 PRECURSOR(FGF-22); the human gene sequence reference: NM_(—)020637 ACCESSION:NM_(—)020637 NID: gi 10190671 ref NM_(—)020637.1 Homo sapiens fibroblastgrowth factor 22 (FGF22); the human protein sequence corresponds toreference: Q9HCT0 ACCESSION: Q9HCT0 NID: Homo sapiens (Human).FIBROBLAST GROWTH FACTOR-22 PRECURSOR (FGF-22).

The mouse gene of interest is Fgf22 (fibroblast growth factor 22),ortholog of human FGF22. Aliases include FGF-22 and 2210414E06Rik.

FGF22 is a secreted protein that functions as a signal-transducingligand. The protein consists of a signal peptide and a fibroblast growthfactor (FGF) domain and is capable of binding with FGF receptor 2(Umemori et al, Cell 118(2):257-70 (2004); Boilly et al., CytokineGrowth Factor Rev 11(4):295-302 (2000); Eriksson et al., Proc Natl AcadSci USA 88(8):3441-5 (1991); Murzin et al., J Mol Biol 223(2):531-43(1992)). FGF22 is expressed in skin epithelium and the inner root sheathof the hair follicle, where it likely plays a role in hair developmentand cutaneous development and repair (Nakatake et al, Biochim BiophysActa 1517(3):460-3 (2001); Beyer et al, Exp Cell Res 287(2):228-36(2003)); Wilkie et al, Curr Biol 5(5):500-7 (1995)). FGF22 is alsoexpressed in tongue and in brain, where it plays a role in presynapticorganization (Umemori et al, Cell 118(2):257-70 (2004)).

Targeted or gene trap mutations are generated in strain129SvEv^(Brd)-derived embryonic stem (ES) cells. The chimeric mice arebred to C57BL/6J albino mice to generate F1 heterozygous animals. Theseprogeny are intercrossed to generate F2 wild type, heterozygous, andhomozygous mutant progeny. On rare occasions, for example when very fewF1 mice are obtained from the chimera, F1 heterozygous mice are crossedto 129SvEv^(Brd)/C57 hybrid mice to yield additional heterozygousanimals for the intercross to generate the F2 mice. Level I phenotypicanalysis is performed on mice from this generation

wt het hom Total Observed 19 34 16 69 Expected 17.25 34.5 17.25 69Chi-Sq. = 2.31 Significance = 0.31505755 (hom/n) = 0.25 Avg. Litter Size= 10Mutation InformationMutation Type Homologous Recombination (standard)Description: Coding exons 1 through 3 were targeted (NCBI accessionNM_(—)023304.1).1. Wild-type Expression Panel: Expression of the target gene wasdetected in brain, spinal cord, eye, and thymus among 13 adult tissuesamples tested by RT-PCR.2. QC Expression: QC Images: Disruption of the target gene was confirmedby Southern hybridization analysis.

48.38.1. Phenotypic Analysis (for Disrupted Gene: DNA108912-2680(UNQ2500)

(a) Overall Phenotypic Summary:

Mutation of the gene encoding the ortholog of human fibroblast growthfactor 22 (FGF22) resulted in a decreased percentage of natural killer(NK) cells in the peripheral blood of (−/−) mice. Disruption of thetarget gene was confirmed by Southern hybridization analysis.

(b) Immunology Phenotypic Analysis

Immune related and inflammatory diseases are the manifestation orconsequence of fairly complex, often multiple interconnected biologicalpathways which in normal physiology are critical to respond to insult orinjury, initiate repair from insult or injury, and mount innate andacquired defense against foreign organisms. Disease or pathology occurswhen these normal physiological pathways cause additional insult orinjury either as directly related to the intensity of the response, as aconsequence of abnormal regulation or excessive stimulation, as areaction to self, or as a combination of these.

Though the genesis of these diseases often involves multistep pathwaysand often multiple different biological systems/pathways, interventionat critical points in one or more of these pathways can have anameliorative or therapeutic effect. Therapeutic intervention can occurby either antagonism of a detrimental process/pathway or stimulation ofa beneficial process/pathway.

T lymphocytes (T cells) are an important component of a mammalian immuneresponse. T cells recognize antigens which are associated with aself-molecule encoded by genes within the major histocompatibilitycomplex (MHC). The antigen may be displayed together with MHC moleculeson the surface of antigen presenting cells, virus infected cells, cancercells, grafts, etc. The T cell system eliminates these altered cellswhich pose a health threat to the host mammal. T cells include helper Tcells and cytotoxic T cells. Helper T cells proliferate extensivelyfollowing recognition of an antigen-MHC complex on an antigen presentingcell. Helper T cells also secrete a variety of cytokines, i.e.,lymphokines, which play a central role in the activation of B cells,cytotoxic T cells and a variety of other cells which participate in theimmune response.

In many immune responses, inflammatory cells infiltrate the site ofinjury or infection. The migrating cells may be neutrophilic,eosinophilic, monocytic or lymphocytic as can be determined byhistological examination of the affected tissues. Current Protocols inImmunology, ed. John E. Coligan, 1994, John Wiley & Sons, Inc.

Many immune related diseases are known and have been extensivelystudied. Such diseases include immune-mediated inflammatory diseases(such as rheumatoid arthritis, immune mediated renal disease,hepatobiliary diseases, inflammatory bowel disease (IBD), psoriasis, andasthma), non-immune-mediated inflammatory diseases, infectious diseases,immunodeficiency diseases, neoplasia, and graft rejection, etc. In thearea of immunology, targets were identified for the treatment ofinflammation and inflammatory disorders.

In the area of immunology, targets have been identified herein for thetreatment of inflammation and inflammatory disorders. Immune relateddiseases, in one instance, could be treated by suppressing the immuneresponse. Using neutralizing antibodies that inhibit molecules havingimmune stimulatory activity would be beneficial in the treatment ofimmune-mediated and inflammatory diseases. Molecules which inhibit theimmune response can be utilized (proteins directly or via the use ofantibody agonists) to inhibit the immune response and thus ameliorateimmune related disease.

The following test was performed:

Fluorescence-Activated Cell-Sorting (FACS) Analysis

Procedure:

FACS analysis of immune cell composition from peripheral blood wasperformed including CD4, CD8 and T cell receptor to evaluate Tlymphocytes, CD19 for B lymphocytes, CD45 as a leukocyte marker and panNK for natural killer cells. The FACS analysis was carried out on 2 wildtype and 6 homozygous mice and included cells derived from thymus,spleen, bone marrow and lymph node.

In these studies, analyzed cells were isolated from thymus, peripheralblood, spleen, bone marrow and lymph nodes. Flow cytometry was designedto determine the relative proportions of CD4 and CD8 positive T cells, Bcells, NK cells and monocytes in the mononuclear cell population. ABecton-Dickinson FACS Calibur 3-laser FACS machine was used to assessimmune status. For Phenotypic Assays and Screening, this machine recordsCD4+/CD8−, CD8+/CD4−, NK, B cell and monocyte numbers in addition to theCD4+/CD8+ ratio.

The mononuclear cell profile was derived by staining a single sample oflysed peripheral blood from each mouse with a panel of sixlineage-specific antibodies: CD45 PerCP, anti-TCRb APC, CD4 PE, CD8FITC, pan-NK PE, and CD19 FITC. The two FITC and PE labeled antibodiesstain mutually exclusive cell types. The samples were analyzed using aBecton Dickinson FACS Calibur flow cytometer with CellQuest software.

Results:

FACS3: The (−/−) mice exhibited an altered distribution of leukocytesubsets in the peripheral blood, characterized by a decreased meanpercentage of natural killer cells when compared with that of their(+/+) littermates and the historical mean.

In summary, the FACS results indicate that the homozygous mutant micehave an impaired immune system, especially in view of the decreased meanpercentage of natural killer cells which is an indicator of a negativephenotype associated with knocking out the DNA108912-2680 gene whichencodes PRO5800 polypeptides. Natural killer cells are the first line ofdefense to viral infection since these cells have been implicated inviral immunity and in defense against tumors. Natural killer cells or NKcells act as effectors in antibody-dependent cell-mediated cytotoxicityand have been identified by their ability to kill certain lymphoid tumorcell lines in vitro without the need for prior immunization oractivation. However, their known function in host defense is in theearly phases of infection with several intracellular pathogens,particularly herpes viruses. Thus, PRO5800 polypeptides and agoniststhereof would be important for a healthy immune system and would beuseful in stimulating the immune system particularly during viralinfections.

48.39. Generation and Analysis of Mice Comprising DNA100276-2684(UNQ2504) Gene Disruptions

In these knockout experiments, the gene encoding PRO5993 polypeptides(designated as DNA100276-2684) (UNQ2504) was disrupted. The genespecific information for these studies is as follows: the mutated mousegene corresponds to nucleotide reference:AF358257 ACCESSION:AF358257NID: 17529688 Mus musculus C21orf63 protein (C21orf63); proteinreference: P58659 ACCESSION:P58659 NID: Mus musculus (Mouse). PROTEINC21ORF63 HOMOLOG PRECURSOR; the human gene sequence reference:NM_(—)058187 Homo sapiens chromosome 21 open reading frame 63(C21orf63); the human protein sequence corresponds to reference: P58658ACCESSION:P58658 NID: Homo sapiens (Human). PROTEIN C21ORF63 PRECURSOR(PROTEIN PRED34) (SUE21).

The mouse gene of interest is RIKEN cDNA 4931408A02 gene, ortholog ofhuman C21orf63 (chromosome 21 open reading frame 63). Aliases include1700092M14Rik, B18, SUE21, and PRED34.

C21orf63 is a putative plasma membrane protein (Clark et al, Genome Res13(10):2265-70 (2003)) that may function as a cell adhesion molecule orsignal-transducing receptor. The protein contains two extracellulargalactose-binding lectin domains (PFAM accession PF02140), atransmembrane segment, and a 100-amino acid cytoplasmic segment.

Targeted or gene trap mutations are generated in strain129SvEv^(Brd)-derived embryonic stem (ES) cells. The chimeric mice arebred to C57BL/6J albino mice to generate F1 heterozygous animals. Theseprogeny are intercrossed to generate F2 wild type, heterozygous, andhomozygous mutant progeny. On rare occasions, for example when very fewF1 mice are obtained from the chimera, F1 heterozygous mice are crossedto 129SvEv^(Brd)/C57 hybrid mice to yield additional heterozygousanimals for the intercross to generate the F2 mice. Level I phenotypicanalysis is performed on mice from this generation

wt het hom Total Observed 17 45 24 86 Expected 21.5 43 21.5 86 Chi-Sq. =1.16 Significance = 0.5598984 (hom/n) = 0.27 Avg. Litter Size = 9Mutation InformationMutation Type Homologous Recombination (standard)Description: Coding exon 1 was targeted (NCBI accession AF358257).1. Wild-type Expression Panel: Expression of the target gene wasdetected in embryonic stem (ES) cells and in all 13 adult tissue samplestested by RT-PCR, except skeletal muscle and bone.2. QC Expression: QC Images: Disruption of the target gene was confirmedby Southern hybridization analysis.48.39.1. Phenotypic Analysis (for Disrupted Gene: DNA100276-2684(UNQ2504)

(a) Overall Phenotypic Summary:

UNQ2504, DNA100276 Mutation of the gene encoding the ortholog of humanchromosome 21 open reading frame 63 (C21orf63) resulted in resistance tothe pupil dilating drug cyclopentolate hydrochloride in (−/−) mice. Thehomozygous mutant mice exhibited resistance to the dilating drug usedduring fundus examination when compared with that of their wild-typelittermates and the historical mean. In addition, the mutant (−/−) miceshowed decreased lean body mass and decreased latency to respond in hotplate testing. Disruption of the target gene was confirmed by Southernhybridization analysis.

(b) Bone Metabolism & Body Diagnostics: Radiology Phenotypic Analysis

In the area of bone metabolism, targets were identified herein for thetreatment of arthritis, osteoporosis, osteopenia and osteopetrosis aswell as identifying targets that promote bone healing. Tests included:

DEXA for measurement of bone mineral density on femur and vertebra

MicroCT for very high resolution and very high sensitivity measurementsof bone mineral density for both trabecular and cortical bone.

Dexa Analysis—Test Description:

Procedure: A cohort of 4 wild type, 4 heterozygous and 8 homozygous weretested in this assay. Dual Energy X-ray Absorptiometry (DEXA) has beenused successfully to identify changes in bone. Anesthetized animals wereexamined and bone mineral content (BMC), BMC/LBM ratios, volumetric bonemineral density (vBMD), total body BMD, femur BMD and vertebra BMD weremeasured.

The mouse was anesthetized by intraperitoneal injection of Avertin(1.25% 2,2,2,-tribromoethanol, 20 ml/kg body weight), body length andweight were measured, and then the mouse was placed in a prone positionon the platform of the PIXImus™ Densitometer (Lunar Inc.) for a DEXAscan. Using Lunar PIXImus software, the bone mineral density (BMD) andfat composition (% fat) and total tissue mass (TTM) were determined inthe regions of interest (ROI) [i.e., whole body, vertebrae, and bothfemurs].

Results:

DEXA: The (−/−) mice exhibited decreased mean lean body mass whencompared with those of their gender-matched (+/+) littermates and thehistorical means.

Mutant (−/−) mice deficient in the gene encoding PRO5993 polypeptidesshow a phenotype consistent with tissue wasting diseases (decreased leanbody mass). Thus, antagonists or inhibitors of PRO5993 polypeptides orits encoding gene would mimic these metabolic related effects. On theother hand, PRO5993 polypeptides or agonists thereof would be useful inthe prevention and/or treatment of such metabolic disorders as cachexiaor other tissue wasting diseases.

(c) Cardiovascular Phenotypic Analysis:

In the area of cardiovascular biology, phenotypic testing was performedto identify potential targets for the treatment of cardiovascular,endothelial or angiogenic disorders. One such phenotypic test includedoptic fundus photography and angiography to determine the retinalarteriovenous ratio (A/V ratio) in order to flag various eyeabnormalities. An abnormal A/V ratio signals such systemic diseases ordisorders that may be related to the vascular disease of hypertension(and any disease that causes hypertension, e.g. atherosclerosis),diabetes or other ocular diseases corresponding to opthalmologicaldisorders. Such eye abnormalities may include but are not limited to thefollowing: retinal abnormality is retinal dysplasia, variousretinopathies, restenosis, retinal artery obstruction or occlusion;retinal degeneration causing secondary atrophy of the retinalvasculature, retinitis pigmentosa, macular dystrophies, Stargardt'sdisease, congenital stationary night blindness, choroideremia, gyrateatrophy, Leber's congenital amaurosis, retinoschisis disorders, Wagner'ssyndrome, Usher syndromes, Zellweger syndrome, Saldino-Mainzer syndrome,Senior-Loken syndrome, Bardet-Biedl syndrome, Alport's syndrome,Alstom's syndrome, Cockayne's syndrome, dysplaisa spondyloepiphysariacongentia, Flynn-Aird syndrome, Friedreich ataxia, Hallgren syndrome,Marshall syndrome, Albers-Schnoberg disease, Refsum's disease,Kearns-Sayre syndrome, Waardenburg's syndrome, Alagile syndrome,myotonic dystrophy, olivopontocerebellar atrophy, Pierre-Mariedunsdrome, Stickler syndrome, carotinemeia, cystinosis, Wolframsyndrome, Bassen-Kornzweig syndrome, abetalipoproteinemia, incontinentiapigmenti, Batten's disease, mucopolysaccharidoses, homocystinuria, ormannosidosis.

Procedure: A cohort of 4 wild type, 4 heterozygous and 8 homozygous weretested in this assay. Optic fundus photography was performed onconscious animals using a Kowa Genesis small animal fundus cameramodified according to Hawes and coauthors (Hawes et al., 1999 MolecularVision 1999; 5:22). Intra-peritoneal injection of fluorescein permittedthe acquisition of direct light fundus images and fluorescent angiogramsfor each examination. In addition to direct opthalmological changes,this test can detect retinal changes associated with systemic diseasessuch as diabetes and atherosclerosis or other retinal abnormalities.Pictures were provided of the optic fundus under normal light. Theangiographic pictures allowed examination of the arteries and veins ofthe eye. In addition an artery to vein (A/V) ratio was determined forthe eye.

Opthalmology analysis was performed on generated F2 wild type,heterozygous, and homozygous mutant progeny using the protocol describedabove. Specifically, the A/V ratio was measured and calculated accordingto the fundus images with Kowa COMIT+ software. This test takes colorphotographs through a dilated pupil: the images help in detecting andclassifying many diseases. The artery to vein ratio (A/V) is the ratioof the artery diameter to the vein diameter (measured before thebifurcation of the vessels). Many diseases will influence the ratio,i.e., diabetes, cardiovascular disorders, papilledema, optic atrophy orother eye abnormalities such as retinal degeneration (known as retinitispigmentosa) or retinal dysplasia, vision problems or blindness. Thus,phenotypic observations which result in an increased artery-to-veinratio in homozygous (−/−) and heterozygous (+/−) mutant progeny comparedto wild-type (+/+) littermates would be indicative of such pathologicalconditions.

Results:

Fundus: The (−/−) mice exhibited resistance to the pupil dilating drugcyclopentolate hydrochloride. Only 4 males were examined; of these, onlytwo images are interpretable; the pupils did not dilate normally inthese animals. Two images show only cloudiness. One of the animals hadcloudiness on fundus exam and was also reported to have “white spots” onthe eyes. Functional observation battery testing resulted in onewild-type (+/+) mouse and two homozygous (−/−) mice having noted eyechanges including small squinty eyes; one (−/−) mouse showed white spotson squinty eyes. The (−/−) mice exhibited body tremors and reducedexploratory behavior. Thus, the mutant (−/−) mice examined showed someretinal abnormalities which could be related to corneal changes and/orcataract formation.

(d) Phenotypic Analysis: CNS/Neurology

In the area of neurology, analysis focused herein on identifying in vivovalidated targets for the treatment of neurological and psychiatricdisorders including depression, generalized anxiety disorders, attentiondeficit hyperactivity disorder, obsessive compulsive disorder,schizophrenia, cognitive disorders, hyperalgesia and sensory disorders.Neurological disorders include the category defined as “anxietydisorders” which include but are not limited to: mild to moderateanxiety, anxiety disorder due to a general medical condition, anxietydisorder not otherwise specified, generalized anxiety disorder, panicattack, panic disorder with agoraphobia, panic disorder withoutagoraphobia, posttraumatic stress disorder, social phobia, specificphobia, substance-induced anxiety disorder, acute alcohol withdrawal,obsessive compulsive disorder, agoraphobia, bipolar disorder I or II,bipolar disorder not otherwise specified, cyclothymic disorder,depressive disorder, major depressive disorder, mood disorder,substance-induced mood disorder. In addition, anxiety disorders mayapply to personality disorders including but not limited to thefollowing types: paranoid, antisocial, avoidant behavior, borderlinepersonality disorders, dependent, histronic, narcissistic,obsessive-compulsive, schizoid, and schizotypal.

Procedure:

Behavioral screens were performed on a cohort of 4 wild type, 4heterozygous and 8 homozygous mutant mice. All behavioral tests weredone between 12 and 16 weeks of age unless reduced viabilitynecessitates earlier testing. These tests included open field to measureanxiety, activity levels and exploration.

Hot Plate Testing

Test Description: The hot plate test for nociception is carried out byplacing each mouse on a small enclosed 55° C. hot plate. Latency to ahindlimb response (lick, shake, or jump) is recorded, with a maximumtime on the hot plate of 30 sec. Each animal is tested once.

Results:

Hot Plate: The (−/−) mice exhibited a decreased latency to respond,suggesting an increased sensitivity to acute pain in the mutants.

48.40. Generation and Analysis of Mice Comprising DNA96860-2700(UNQ2524) Gene Disruptions

In these knockout experiments, the gene encoding PRO6017 polypeptides(designated as DNA96860-2700) (UNQ2524) was disrupted. The gene specificinformation for these studies is as follows: the mutated mouse genecorresponds to nucleotide reference: XM_(—)356118 PREDICTED: Musmusculus gene model 1109, (NCBI) (Gm1109); protein reference:XP_(—)356118 PREDICTED: similar to G-protein coupled receptor 114 [Musmusculus]; the human gene sequence reference: NM_(—)153837 ACCESSION:NM_(—)153837 NID: gi 24475870 ref NM_(—)153837.1 Homo sapiens Gprotein-coupled receptor 114 (GPR114); the human protein sequencecorresponds to reference: NP_(—)722579 G-protein coupled receptor 114[Homo sapiens] gi|22749621|gb|AAH32401.1| G-protein coupled receptor 114[Homo sapiens].

The mouse gene of interest is Gpr114 (G protein-coupled receptor 114),ortholog of human GPR114. Aliases include PGR27 and Gm1109.

GPR114 is a G protein-coupled receptor of the secretin family(Fredriksson et al, FEBS Lett 531(3):407-14 (2002); Bjarnadottir et al,Genomics 84(1):23-33 (2004)). Members of this family generally consistof a large N-terminal segment, a G protein-coupled receptor proteolyticsite (GPS) domain (SMART accession SM00303), and a secretin familyseven-transmembrane receptor domain (Pfam accession PF00002). Secretinfamily G protein-coupled receptors include secretin, calcitonin, andvasoactive intestinal peptide receptors, which activate adenyl cyclaseor phospholipase C (Pfam accession PF00002).

Targeted or gene trap mutations are generated in strain129SvEv^(Brd)-derived embryonic stem (ES) cells. The chimeric mice arebred to C57BL/6J albino mice to generate F1 heterozygous animals. Theseprogeny are intercrossed to generate F2 wild type, heterozygous, andhomozygous mutant progeny. On rare occasions, for example when very fewF1 mice are obtained from the chimera, F1 heterozygous mice are crossedto 129SvEv^(Brd)/C57 hybrid mice to yield additional heterozygousanimals for the intercross to generate the F2 mice. Level I phenotypicanalysis is performed on mice from this generation

wt het hom Total Observed 19 38 17 74 Expected 18.5 37 18.5 74 Chi-Sq. =1.16 Significance = 0.5598984 (hom/n) = 0.24 Avg. Litter Size = 10Mutation InformationMutation Type: Homologous Recombination (standard)Description: Coding exons 4 through 7 were targeted (NCBI accessionXM_(—)356118.2).1. Wild-type Expression Panel: Expression of the target gene wasdetected in all 13 adult tissue samples tested by RT-PCR, except brain;lung; liver; skeletal muscle; bone; stomach, small intestine, and colon;and adipose.2. QC Expression: QC Images: Disruption of the target gene was confirmedby Southern hybridization analysis.

48.40.1. Phenotypic Analysis (for Disrupted Gene: DNA96860-2700(UNQ2524)

(a) Overall Phenotypic Summary:

Mutation of the gene encoding the ortholog of human G protein-coupledreceptor 114 (GPR114) resulted in the (−/−) mice exhibiting decreasedlean body mass and decreased bone mineral density measurements. Genedisruption was confirmed by Southern blot.

(b) Bone Metabolism & Body Diagnostics: Radiology Phenotypic Analysis

In the area of bone metabolism, targets were identified herein for thetreatment of arthritis, osteoporosis, osteopenia and osteopetrosis aswell as identifying targets that promote bone healing. Tests included:

DEXA for measurement of bone mineral density on femur and vertebra

MicroCT for very high resolution and very high sensitivity measurementsof bone mineral density for both trabecular and cortical bone.

Dexa Analysis—Test Description:

Procedure: A cohort of 4 wild type, 4 heterozygous and 8 homozygous weretested in this assay. Dual Energy X-ray Absorptiometry (DEXA) has beenused successfully to identify changes in bone. Anesthetized animals wereexamined and bone mineral content (BMC), BMC/LBM ratios, volumetric bonemineral density (vBMD), total body BMD, femur BMD and vertebra BMD weremeasured.

The mouse was anesthetized by intraperitoneal injection of Avertin(1.25% 2,2,2,-tribromoethanol, 20 ml/kg body weight), body length andweight were measured, and then the mouse was placed in a prone positionon the platform of the PIXImus™ Densitometer (Lunar Inc.) for a DEXAscan. Using Lunar PIXImus software, the bone mineral density (BMD) andfat composition (% fat) and total tissue mass (TTM) were determined inthe regions of interest (ROI) [i.e., whole body, vertebrae, and bothfemurs].

Bone MicroCT Analysis:

Procedure: MicroCT was also used to get very sensitive measurements ofBMD. One vertebra and 1 femur were taken from a cohort of 4 wild typeand 8 homozygous mice. Measurements were taken of lumbar 5 vertebratrabecular bone volume, trabecular thickness, connectivity density andmidshaft femur total bone area and cortical thickness. The μCT40 scansprovided detailed information on bone mass and architecture. Multiplebones were placed into sample holders and scanned automatically.Instrument software was used to select regions of interest for analysis.Trabecular bone parameters were analyzed in the fifth lumbar vertebrae(LV5) at 16 micrometer resolution and cortical bone parameters wereanalyzed in the femur midshaft at a resolution of 20 micrometers.

Results:

DEXA: The male (−/−) mice exhibited decreased mean lean body mass whencompared with that of their gender-matched (+/+) littermates and thehistorical mean. The male (−/−) mice also exhibited decreased mean bonemineral content and density-related measurements.Micro CT: The male (−/−) mice exhibited decreased mean femoral mid-shaftcross-sectional area when compared with that of their gender-matched(+/+) littermates and the historical mean.

Mutant (−/−) mice deficient in the gene encoding PRO6017 polypeptidesshow a phenotype consistent with tissue wasting diseases (decreased leanbody mass). Thus, antagonists or inhibitors of PRO6017 polypeptides orits encoding gene would mimic these metabolic related effects. On theother hand, PRO6017 polypeptides or agonists thereof would be useful inthe prevention and/or treatment of such metabolic disorders as cachexiaor other tissue wasting diseases.

In addition, the (−/−) mice analyzed by DEXA exhibited decreased bonemeasurements and decreased body mass measurements when compared withtheir (+/+) littermates, suggestive of abnormal bone disorders. Inaddition, the decreased lean body mass is indicative of a metabolicdisorder related to growth retardation and tissue wasting disorders. Thenegative bone phenotype indicates that PRO6017 polypeptides or agoniststhereof would be useful for maintaining bone homeostasis in addition tonormal growth development. In addition, PRO6017 polypeptides would beuseful in bone healing or for the treatment of arthritis orosteoporosis, whereas antagonists (or inhibitors) of PRO6017polypeptides or its encoding gene would lead to abnormal or pathologicalbone disorders including inflammatory diseases associated with abnormalbone metabolism including arthritis, osteoporosis and osteopenia.

48.41. Generation and Analysis of Mice Comprising DNA96883-2745(UNQ2784) Gene Disruptions

In these knockout experiments, the gene encoding PRO7174 polypeptides(designated as DNA96883-2745) (UNQ2784) was disrupted. The gene specificinformation for these studies is as follows: the mutated mouse genecorresponds to nucleotide reference: NM_(—)199222 Mus musculus cDNAsequence BC020188 (BC020188); protein reference:Q8VCD3 ACCESSION: Q8VCD3NID: Mus musculus (Mouse). ERGIC-53-like protein precursor (Lectin,mannose-binding 1 like); the human gene sequence reference: NM_(—)021819ACCESSION: NM_(—)021819 NID: gi 11141890 ref NM_(—)021819.1 Homo sapienslectin, mannose-binding, 1 like (LMAN1L); the human protein sequencecorresponds to reference: Q9HAT1 ACCESSION: Q9HAT1 NID: Homo sapiens(Human). ERGIC-53-like protein precursor (Lectin, mannose-binding 1like).

The mouse gene of interest is “cDNA sequence BC020188,” ortholog ofhuman LMAN1L (lectin, mannose-binding, 1 like). Aliases include ERGL,CPLX3, CPXIII, FLJ13993, ERGIC-53L, complexin III, and ERGIC-53-likeprotein.

LMAN1L is a putative type I membrane protein, containing a signalpeptide, a leguminous lectin-like domain (Pfam accession PF03388), and atransmembrane segment. The protein likely functions as a cargo receptoror regulator of cargo receptor ERGIC-53 located in the endoplasmicreticulum (ER)-Golgi intermediate compartment. ERGIC-53 binds withmannose-containing glycoproteins and participates in the sorting andtransfer of glycoproteins from the endoplasmic reticulum to the Golgicomplex (Yerushalmi et al, Gene 265(1-2):55-60 (2001); Hauri et al,Biochem Soc Symp 69:73-82 (2002); Schrag et al, Trends Biochem Sci28(1):49-57 (2003)). Like ERGIC-53, LMAN1L is likely located in theER-Golgi intermediate compartment; however, bioinformatic analysessuggest that LMAN1L is an extracellular protein (Clark et al, Genome Res13(10):2265-70 (2003)). LMAN1L is expressed in prostate, cardiac atrium,salivary gland, spleen, and central nervous system and is likely to playa role in glycoprotein folding and secretion (Yerushalmi et al, Gene265(1-2):55-60 (2001); Hauri et al, Biochem Soc Symp 69:73-82 (2002);Schrag et al, Trends Biochem Sci 28(1):49-57 (2003)).

Targeted or gene trap mutations are generated in strain129SvEv^(Brd)-derived embryonic stem (ES) cells. The chimeric mice arebred to C57BL/6J albino mice to generate F1 heterozygous animals. Theseprogeny are intercrossed to generate F2 wild type, heterozygous, andhomozygous mutant progeny. On rare occasions, for example when very fewF1 mice are obtained from the chimera, F1 heterozygous mice are crossedto 129SvEv^(Brd)/C57 hybrid mice to yield additional heterozygousanimals for the intercross to generate the F2 mice. Level I phenotypicanalysis is performed on mice from this generation

wt het hom Total Observed 19 24 15 58 Expected 14.5 29 14.5 58 Chi-Sq. =1.76 Significance = 0.4147829 (hom/n) = 0.26 Avg. Litter Size = 8Mutation InformationMutation Type: Homologous Recombination (standard)Description: Coding exons 2 through 4 were targeted (NCBI accessionNM_(—)021819.1 [human]).1. Wild-type Expression Panel: Expression of the target gene wasdetected only in eye and spleen among the 13 adult tissue samples testedby RT-PCR.2. QC Expression: QC Images: Disruption of the target gene was confirmedby Southern hybridization analysis.

48.41.1. Phenotypic Analysis (for Disrupted Gene: DNA96883-2745(UNQ2784)

(a) Overall Phenotypic Summary:

Mutation of the gene encoding the ortholog of human lectin,mannose-binding, 1 like (LMAN1L) resulted in increased serum glucose aswell as increased mean serum cholesterol levels in both (+/−) and (−/−)mice. Urinalysis showed increased urobilinogen levels. The mutant (−/−)mice exhibited extramedullary hematopoiesis. The mutant (−/−) mice alsoexhibited numerous immunological abnormalities. Circadian testing showeddecreased ambulation counts or hypoactivity in the (−/−) mice. Themutant (−/−) mice also showed decreased trabecular bone volume, numberand connectivity density. Gene disruption was confirmed by Southernblot.

(b) Expression

GeneLogic expression patterns showed high tissue expression in lymph andprostate. A specific signal was observed in the spleen by ISH. Signalstrength was as strong as that observed in prostate carcinoma and wasselective of the broad marginal zones of B cell splenic follicles.UNQ2784 expression and knockout phenotype supports a role in B cellfunction. [See EXAMPLES 57 for protocol]

(c) Immunology Phenotypic Analysis

Immune related and inflammatory diseases are the manifestation orconsequence of fairly complex, often multiple interconnected biologicalpathways which in normal physiology are critical to respond to insult orinjury, initiate repair from insult or injury, and mount innate andacquired defense against foreign organisms. Disease or pathology occurswhen these normal physiological pathways cause additional insult orinjury either as directly related to the intensity of the response, as aconsequence of abnormal regulation or excessive stimulation, as areaction to self, or as a combination of these.

Though the genesis of these diseases often involves multistep pathwaysand often multiple different biological systems/pathways, interventionat critical points in one or more of these pathways can have anameliorative or therapeutic effect. Therapeutic intervention can occurby either antagonism of a detrimental process/pathway or stimulation ofa beneficial process/pathway.

T lymphocytes (T cells) are an important component of a mammalian immuneresponse. T cells recognize antigens which are associated with aself-molecule encoded by genes within the major histocompatibilitycomplex (MHC). The antigen may be displayed together with MHC moleculeson the surface of antigen presenting cells, virus infected cells, cancercells, grafts, etc. The T cell system eliminates these altered cellswhich pose a health threat to the host mammal. T cells include helper Tcells and cytotoxic T cells. Helper T cells proliferate extensivelyfollowing recognition of an antigen-MHC complex on an antigen presentingcell. Helper T cells also secrete a variety of cytokines, i.e.,lymphokines, which play a central role in the activation of B cells,cytotoxic T cells and a variety of other cells which participate in theimmune response.

In many immune responses, inflammatory cells infiltrate the site ofinjury or infection. The migrating cells may be neutrophilic,eosinophilic, monocytic or lymphocytic as can be determined byhistological examination of the affected tissues. Current Protocols inImmunology, ed. John E. Coligan, 1994, John Wiley & Sons, Inc.

Many immune related diseases are known and have been extensivelystudied. Such diseases include immune-mediated inflammatory diseases(such as rheumatoid arthritis, immune mediated renal disease,hepatobiliary diseases, inflammatory bowel disease (IBD), psoriasis, andasthma), non-immune-mediated inflammatory diseases, infectious diseases,immunodeficiency diseases, neoplasia, and graft rejection, etc. In thearea of immunology, targets were identified for the treatment ofinflammation and inflammatory disorders.

In the area of immunology, targets have been identified herein for thetreatment of inflammation and inflammatory disorders. Immune relateddiseases, in one instance, could be treated by suppressing the immuneresponse. Using neutralizing antibodies that inhibit molecules havingimmune stimulatory activity would be beneficial in the treatment ofimmune-mediated and inflammatory diseases. Molecules which inhibit theimmune response can be utilized (proteins directly or via the use ofantibody agonists) to inhibit the immune response and thus ameliorateimmune related disease.

The following tests were performed:

Hematology Analysis:

Test Description: Blood tests are carried out by Abbott's Cell-Dyn3500R, an automated hematology analyzer. Some of its features include afive-part WBC differential. ‘Patient’ reports can cover over 22parameters in all.

Results:

Hematology: The (−/−) mice exhibited an increased mean platelet countwhen compared with that of their (+/+) littermates and the historicalmean.

Thus, mutant mice deficient in the DNA96883-2745 gene resulted in aphenotype related to coagulation disorders. In this regard, inhibitorsor antagonists of PRO7174 polypeptides would be useful in treatingdisorders related to abnormal blood coagulation such as hemophilia.

Fluorescence-Activated Cell-Sorting (FACS) Analysis

Procedure:

FACS analysis of immune cell composition from peripheral blood wasperformed including CD4, CD8 and T cell receptor to evaluate Tlymphocytes, CD19 for B lymphocytes, CD45 as a leukocyte marker and panNK for natural killer cells. The FACS analysis was carried out on 2 wildtype and 6 homozygous mice and included cells derived from thymus,spleen, bone marrow and lymph node.

In these studies, analyzed cells were isolated from thymus, peripheralblood, spleen, bone marrow and lymph nodes. Flow cytometry was designedto determine the relative proportions of CD4 and CD8 positive T cells, Bcells, NK cells and monocytes in the mononuclear cell population. ABecton-Dickinson FACSCalibur 3-laser FACS machine was used to assessimmune status. For Phenotypic Assays and Screening, this machine recordsCD4+/CD8−, CD8+/CD4−, NK, B cell and monocyte numbers in addition to theCD4+/CD8+ ratio.

The mononuclear cell profile was derived by staining a single sample oflysed peripheral blood from each mouse with a panel of sixlineage-specific antibodies: CD45 PerCP, anti-TCRb APC, CD4 PE, CD8FITC, pan-NK PE, and CD19 FITC. The two FITC and PE labeled antibodiesstain mutually exclusive cell types. The samples were analyzed using aBecton Dickinson FACSCalibur flow cytometer with CellQuest software.

Results:

Tissue Specific FACS-Project: The (−/−) mice exhibited a decreased Tcell:B cell ratio in lymph node when compared with that of their (+/+)littermates. The (−/−) mice also exhibited increased percentages ofB220+ CD38 Low IgM− and TCRbeta+ CD38+ cells in Peyer's patches. Inaddition, mild-moderate extramedullary hematopoiesis was reported infour homozygous (−/−) mice.

These observations indicate that there is a change of B cell subtypes inPeyer's patches. Also, an increase in B cell number in lymph nodes wasobserved. Thus, it appears that PRO7174 polypeptides acts as a negativeregulator for B cell production and a positive regulator for T cellproduction.

(d) Phenotypic Analysis: Cardiology

In the area of cardiovascular biology, targets were identified hereinfor the treatment of hypertension, atherosclerosis, heart failure,stroke, various coronary artery diseases, dyslipidemias such as highcholesterol (hypercholesterolemia) and elevated serum triglycerides(hypertriglyceridemia), diabetes and/or obesity. The phenotypic testsincluded the measurement of serum cholesterol and triglycerides.

Blood Lipids

Procedure: A cohort of 4 wild type, 4 heterozygous and 8 homozygous micewere tested in this assay. High cholesterol levels and increasedtriglyceride blood levels are recognized risk factors in the developmentof cardiovascular disease and/or diabetes. Measuring blood lipidsfacilitates the finding of biological switches that regulate blood lipidlevels. Inhibition of factors which elevate blood lipid levels may beuseful for reducing the risk for cardiovascular disease. In these bloodchemistry tests, measurements were recorded using the COBAS Integra 400(mfr: Roche).

Results:

Blood Chemistry: The male (+/−) and (−/−) mice exhibited increased meanserum cholesterol when compared with those of their gender-matched (+/+)littermates and the historical means.

As summarized above, the (+/−) and (−/−) mice exhibited increased meanserum cholesterol levels when compared with their gender-matched (+/+)littermates and the historical means. Thus, mutant mice deficient in thePRO7174 gene may serve as a model for cardiovascular disease. PRO7174polypeptides or its encoding gene would be useful in regulating bloodlipids such as cholesterol. Thus, PRO7174 polypeptides or agoniststhereof would be useful in the treatment of such cardiovascular diseasesas hypertension, atherosclerosis, heart failure, stroke, variouscoronary diseases, hypercholesterolemia, diabetes and/or obesity.

(e) Phenotypic Analysis: Metabolism—Blood Chemistry/Urinalysis

In the area of metabolism, targets may be identified for the treatmentof diabetes. Blood chemistry phenotypic analysis includes blood glucosemeasurements. The COBAS Integra 400 (mfr: Roche) was used for runningblood chemistry tests on the mice. In addition to measuring bloodglucose levels the following blood chemistry tests are also routinelyperformed: Alkaline Phosphatase; Alanine Amino-Transferase; Albumin;Bilirubin; Phosphorous; Creatinine; BUN=Blood Urea Nitrogen; Calcium;Uric Acid; Sodium; Potassium; and Chloride. In the area of metabolism,targets may be identified for the treatment of diabetes. Blood chemistryphenotypic analysis includes glucose tolerance tests to measure insulinsensitivity and changes in glucose metabolism. Abnormal glucosetolerance test results may indicate but may not be limited to thefollowing disorders or conditions: Diabetes Type 1 and Type 2, SyndromeX, various cardiovascular diseases and/or obesity.

Results:

Both the male and female (+/−) and (−/−) mice exhibited increased meanserum glucose levels (˜2SD above the historic mean) compared to theirwild-type (+/+) littermates and the historical mean.

Thus, the mutant (+/−) and (−/−) mice exhibited hyperglycemia which isassociated with an altered glucose metabolism or diabetes. PRO7174polypeptides or agonists thereof would be useful in maintaining normalglucose levels/metabolism and possibly useful in the treatment ofdiabetes.

In addition to the elevated mean serum glucose levels in theheterozygous and homozygous mice, the male and female mutant mice alsoshowed grossly elevated levels of urinary urobilinogen (˜20 foldincrease in two out of four heterozygous (+/−) mice and 3 out of 4homozygous (−/−) mice). Serum bilirubin was normal. These results couldbe a function of an abnormal urine possibly associated with kidneydysfunction.

(f) Phenotypic Analysis: CNS/Neurology

In the area of neurology, analysis focused herein on identifying in vivovalidated targets for the treatment of neurological and psychiatricdisorders including depression, generalized anxiety disorders, attentiondeficit hyperactivity disorder, obsessive compulsive disorder,schizophrenia, cognitive disorders, hyperalgesia and sensory disorders.Neurological disorders include the category defined as “anxietydisorders” which include but are not limited to: mild to moderateanxiety, anxiety disorder due to a general medical condition, anxietydisorder not otherwise specified, generalized anxiety disorder, panicattack, panic disorder with agoraphobia, panic disorder withoutagoraphobia, posttraumatic stress disorder, social phobia, specificphobia, substance-induced anxiety disorder, acute alcohol withdrawal,obsessive compulsive disorder, agoraphobia, bipolar disorder I or II,bipolar disorder not otherwise specified, cyclothymic disorder,depressive disorder, major depressive disorder, mood disorder,substance-induced mood disorder. In addition, anxiety disorders mayapply to personality disorders including but not limited to thefollowing types: paranoid, antisocial, avoidant behavior, borderlinepersonality disorders, dependent, histronic, narcissistic,obsessive-compulsive, schizoid, and schizotypal.

Procedure:

Behavioral screens were performed on a cohort of 4 wild type, 4heterozygous and 8 homozygous mutant mice. All behavioral tests weredone between 12 and 16 weeks of age unless reduced viabilitynecessitates earlier testing. These tests included open field to measureanxiety, activity levels and exploration.

Circadian Test Description:

Female mice are individually housed at 4 pm on the first day of testingin 48.2 cm×26.5 cm home cages and administered food and water adlibitum. Animals are exposed to a 12-hour light/dark cycle with lightsturning on at 7 am and turning off at 7 pm. The system software recordsthe number of beam interruptions caused by the animal's movements, withbeam breaks automatically divided into ambulations. Activity is recordedin 60, one-hour intervals during the three-day test. Data generated aredisplayed by median activity levels recorded for each hour (circadianrhythm) and median total activity during each light/dark cycle(locomotor activity) over the three-day testing period.

Results:

The (−/−) mice exhibited decreased ambulatory counts (hypoactivity)during the 1-hour habituation period and both light periods of home-cageactivity testing when compared with their gender-matched (+/+)littermates and the historical mean. These results are consistent withlethargy or depressive disorders. Antagonists or inhibitors of PRO7174polypeptides or the PRO7174 encoding gene would be expected to mimicthis behavior. Likewise, PRO7174 polypeptides or agonists thereof, wouldbe useful in the treatment of such neurological disorders includingdepressive disorders or other decreased anxiety-like symptoms such aslethargy, cognitive disorders, hyperalgesia and sensory disorders.

(g) Bone Metabolism & Body Diagnostics: Radiology Phenotypic Analysis

In the area of bone metabolism, targets were identified herein for thetreatment of arthritis, osteoporosis, osteopenia and osteopetrosis aswell as identifying targets that promote bone healing. Tests included:

DEXA for measurement of bone mineral density on femur and vertebra

MicroCT for very high resolution and very high sensitivity measurementsof bone mineral density for both trabecular and cortical bone.

Dexa Analysis—Test Description:

Procedure: A cohort of 4 wild type, 4 heterozygous and 8 homozygous micewere tested in this assay. Dual Energy X-ray Absorptiometry (DEXA) hasbeen used successfully to identify changes in bone. Anesthetized animalswere examined and bone mineral content (BMC), BMC/LBM ratios, volumetricbone mineral density (vBMD), total body BMD, femur BMD and vertebra BMDwere measured.

The mouse was anesthetized by intraperitoneal injection of Avertin(1.25% 2,2,2,-tribromoethanol, 20 ml/kg body weight), body length andweight were measured, and then the mouse was placed in a prone positionon the platform of the PIXImus™ Densitometer (Lunar Inc.) for a DEXAscan. Using Lunar PIXImus software, the bone mineral density (BMD) andfat composition (% fat) and total tissue mass (TTM) were determined inthe regions of interest (ROI) [i.e., whole body, vertebrae, and bothfemurs].

Bone MicroCT Analysis:

Procedure: MicroCT was also used to get very sensitive measurements ofBMD. One vertebra and 1 femur were taken from a cohort of 4 wild typeand 8 homozygous mice. Measurements were taken of lumbar 5 vertebratrabecular bone volume, trabecular thickness, connectivity density andmidshaft femur total bone area and cortical thickness. The μCT40 scansprovided detailed information on bone mass and architecture. Multiplebones were placed into sample holders and scanned automatically.Instrument software was used to select regions of interest for analysis.Trabecular bone parameters were analyzed in the fifth lumbar vertebrae(LV5) at 16 micrometer resolution and cortical bone parameters wereanalyzed in the femur midshaft at a resolution of 20 micrometers.

Results:

Micro CT: The male (−/−) mice exhibited decreased mean vertebraltrabecular bone volume, number, and connectivity density when comparedwith that of their gender-matched (+/+) littermates and the historicalmeans.

The (−/−) mice analyzed by Micro CT analysis exhibited decreased bonemeasurements when compared with their (+/+) littermates, suggestive ofabnormal bone disorders. The (−/−) mice exhibited a negative bonephenotype with abnormal and decreased bone measurements reflective ofbone metabolic disorders. The negative bone phenotype indicates thatPRO7174 polypeptides or agonists thereof would be useful for maintainingbone homeostasis. In addition, PRO7174 polypeptides would be useful inbone healing or for the treatment of arthritis or osteoporosis, whereasantagonists (or inhibitors) of PRO7174 polypeptides or its encoding genewould lead to abnormal or pathological bone disorders includinginflammatory diseases associated with abnormal bone metabolism includingarthritis, osteoporosis and osteopenia.

48.42. Generation and Analysis of Mice Comprising DNA136110-2763(UNQ3003) Gene Disruptions

In these knockout experiments, the gene encoding PRO9744 polypeptides(designated as DNA136110-2763) (UNQ3003) was disrupted. The genespecific information for these studies is as follows: the mutated mousegene corresponds to nucleotide reference: NM_(—)022416 Mus musculusserine/threonine kinase 32B (Stk32b); protein reference: Q9JJX8 Q9JJX8Q9JJX8 SERINE/THREONINE PROTEIN KINASE; the human gene sequencereference: NM_(—)018401 Homo sapiens serine/threonine kinase 32B(STK32B); the human protein sequence corresponds to reference: Q9NY57Q9NY57 Q9NY57 SERINE/THREONINE PROTEIN KINASE.

The mouse gene of interest is Stk32b (serine/threonine kinase 32B),ortholog of human STK32B. Aliases include STKG6, Stk32, YANK2,2510009F08Rik, HSA250839, and serine threonine kinase 32.

STK32B is a putative cytosolic serine/threonine protein kinase,containing a serine/threonine protein kinase catalytic domain (SMARTaccession SM00220). Bioinformatic analysis suggests that STK32B may bean extracellular protein (Clark et al, Genome Res 13(10):2265-70(2003)).

Targeted or gene trap mutations are generated in strain129SvEv^(Brd)-derived embryonic stem (ES) cells. The chimeric mice arebred to C57BL/6J albino mice to generate F1 heterozygous animals. Theseprogeny are intercrossed to generate F2 wild type, heterozygous, andhomozygous mutant progeny. On rare occasions, for example when very fewF1 mice are obtained from the chimera, F1 heterozygous mice are crossedto 129SvEv^(Brd)/C57 hybrid mice to yield additional heterozygousanimals for the intercross to generate the F2 mice. Level I phenotypicanalysis is performed on mice from this generation

wt het hom Total Observed 15 38 19 72 Expected 18 36 18 72 Chi-Sq. =0.44 Significance = 0.8025188 (hom/n) = 0.25 Avg. Litter Size = 8Mutation InformationMutation Type Homologous Recombination (standard)Description: Coding exon 3 was targeted (NCBI accession NM_(—)022416.1).1. Wild-type Expression Panel: Expression of the target gene wasdetected in all 26 adult tissue samples tested by RT-PCR, except spleen,lung, liver, skeletal muscle, bone, adipose, asthmatic lung, LPS liver,blood, aortic tree and skin fibroblast.2. QC Expression: QC Images: Disruption of the target gene was confirmedby Southern hybridization analysis.

48.42.1. Phenotypic Analysis (for Disrupted Gene: DNA136110-2763(UNQ3003)

(a) Overall Phenotypic Summary:

Mutation of the gene encoding the ortholog of human serine/threoninekinase 32B (STK32B) resulted in the (−/−) mice exhibiting increased meanserum triglyceride levels. Gene disruption was confirmed by Southernblot.

(b) Phenotypic Analysis: Cardiology

In the area of cardiovascular biology, targets were identified hereinfor the treatment of hypertension, atherosclerosis, heart failure,stroke, various coronary artery diseases, dyslipidemias such as highcholesterol (hypercholesterolemia) and elevated serum triglycerides(hypertriglyceridemia), diabetes and/or obesity. The phenotypic testsincluded the measurement of serum cholesterol and triglycerides.

Blood Lipids

Procedure: A cohort of 4 wild type, 4 heterozygous and 8 homozygous micewere tested in this assay. High cholesterol levels and increasedtriglyceride blood levels are recognized risk factors in the developmentof cardiovascular disease and/or diabetes. Measuring blood lipidsfacilitates the finding of biological switches that regulate blood lipidlevels. Inhibition of factors which elevate blood lipid levels may beuseful for reducing the risk for cardiovascular disease. In these bloodchemistry tests, measurements were recorded using the COBAS Integra 400(mfr: Roche).

Results:

Blood Chemistry: Both the male and female (−/−) mice exhibited increasedmean serum triglyceride levels when compared with those of theirgender-matched (+/+) littermates and the historical means.

As summarized above, the (−/−) mice exhibited notably increased meanserum triglyceride levels when compared with their gender-matched (+/+)littermates and the historical means. Thus, mutant mice deficient in thePRO9744 gene may serve as a model for cardiovascular disease. PRO9744polypeptides or its encoding gene would be useful in regulating bloodlipids such as triglycerides. Thus, PRO9744 polypeptides or agoniststhereof would be useful in the treatment of such cardiovascular diseasesas hypertension, atherosclerosis, heart failure, stroke, variouscoronary diseases, hypertriglyceridemia, diabetes and/or obesity.

48.43. Generation and Analysis of Mice Comprising DNA108725-2766(UNQ3023) Gene Disruptions

In these knockout experiments, the gene encoding PRO9821 polypeptides(designated as DNA108725-2766) (UNQ3023) was disrupted. The genespecific information for these studies is as follows: the mutated mousegene corresponds to nucleotide reference: NM_(—)177139 Mus musculusRIKEN cDNA E130115E03 gene (E130115E03Rik); protein reference:Q8BPP5ACCESSION: Q8BPP5 NID: Mus musculus (Mouse). Protein UNQ3023/PRO9821precursor; the human gene sequence reference: NM_(—)194317 Homo sapienshypothetical protein MGC52057 (MGC52057); the human protein sequencecorresponds to reference: Q86Y78 ACCESSION: Q86Y78 NID: Homo sapiens(Human). Protein UNQ3023/PRO9821 precursor.

The mouse gene of interest is RIKEN cDNA E130115E03 gene, ortholog ofhuman hypothetical protein MGC52057.

Hypothetical protein MGC52057 is a putative extracellular protein (Clarket al, Genome Res 13(10):2265-70 (2003)) consisting of a signal peptideand an Ly-6 antigen/uPA receptor-like (LU) domain (SMART accessionSM00134). Proteins with similar domain organization include CD59antigen, which protects cells from complement-mediated lysis, and LY6D,which is involved in cell-cell adhesion in keratinocytes (Brakenhoff etal, J Cell Biol 129(6):1677-89 (1995); Clayton et al, Eur J Immunol33(2):522-31 (2003)).

Targeted or gene trap mutations are generated in strain129SvEv^(Brd)-derived embryonic stem (ES) cells. The chimeric mice arebred to C57BL/6J albino mice to generate F1 heterozygous animals. Theseprogeny are intercrossed to generate F2 wild type, heterozygous, andhomozygous mutant progeny. On rare occasions, for example when very fewF1 mice are obtained from the chimera, F1 heterozygous mice are crossedto 129SvEv^(Brd)/C57 hybrid mice to yield additional heterozygousanimals for the intercross to generate the F2 mice. Level I phenotypicanalysis is performed on mice from this generation

wt het hom Total Observed 26 29 17 72 Expected 18 36 18 72 Chi-Sq. =4.35 Significance = 0.11360816 (hom/n) = 0.22 Avg. Litter Size = 9Mutation InformationMutation Type Homologous Recombination (standard)Description: Coding exon 1 was targeted (NCBI accession NM_(—)177139.3).1. Wild-type Expression Panel: Expression of the target gene wasdetected in embryonic stem (ES) cells and in all 26 adult tissue samplestested by RT-PCR, except skeletal muscle, adipose, and LPS liver.2. QC Expression: QC Images: Disruption of the target gene was confirmedby Southern hybridization analysis.

48.43.1. Phenotypic Analysis (for Disrupted Gene: DNA108725-2766(UNQ3023)

(a) Overall Phenotypic Summary:

Mutation of the gene encoding the ortholog of human hypothetical proteinMGC52057 resulted in the (−/−) mice exhibiting decreased bone mineralcontent and bone mineral density measurements. UNQ3023 is an unknownprotein and has no immunological phenotype in the (−/−) mice. However,UNQ3023 appears to have a UPAR_LY6 domain in the ECD similar to that inCD59 which is important in the complement pathway. Gene disruption wasconfirmed by Southern blot.

(b) Bone Metabolism & Body Diagnostics: Radiology Phenotypic Analysis

In the area of bone metabolism, targets were identified herein for thetreatment of arthritis, osteoporosis, osteopenia and osteopetrosis aswell as identifying targets that promote bone healing. Tests included:

DEXA for measurement of bone mineral density on femur and vertebra

MicroCT for very high resolution and very high sensitivity measurementsof bone mineral density for both trabecular and cortical bone.

Dexa Analysis—Test Description:

Procedure: A cohort of 4 wild type, 4 heterozygous and 8 homozygous micewere tested in this assay. Dual Energy X-ray Absorptiometry (DEXA) hasbeen used successfully to identify changes in bone. Anesthetized animalswere examined and bone mineral content (BMC), BMC/LBM ratios, volumetricbone mineral density (vBMD), total body BMD, femur BMD and vertebra BMDwere measured.

The mouse was anesthetized by intraperitoneal injection of Avertin(1.25% 2,2,2,-tribromoethanol, 20 ml/kg body weight), body length andweight were measured, and then the mouse was placed in a prone positionon the platform of the PIXImus™ Densitometer (Lunar Inc.) for a DEXAscan. Using Lunar PIXImus software, the bone mineral density (BMD) andfat composition (% fat) and total tissue mass (TTM) were determined inthe regions of interest (ROI) [i.e., whole body, vertebrae, and bothfemurs].

Results:

DEXA: Both the male and female (−/−) mice exhibited decreased bonemineral content, BMC/LBM index and total body bone mineral densitymeasurements when compared with that of their gender-matched (+/+)littermates and the historical means.

The (−/−) mice analyzed by DEXA analysis exhibited decreased bonemeasurements when compared with their (+/+) littermates, suggestive ofabnormal bone disorders. The negative bone phenotype indicates thatPRO9821 polypeptides or agonists thereof would be useful for maintainingbone homeostasis. In addition, PRO9821 polypeptides would be useful inbone healing or for the treatment of arthritis or osteoporosis, whereasantagonists (or inhibitors) of PRO9821 polypeptides or its encoding genewould lead to abnormal or pathological bone disorders includinginflammatory diseases associated with abnormal bone metabolism includingarthritis, osteoporosis and osteopenia.

48.44. Generation and Analysis of Mice Comprising DNA129332-2775(UNQ3037) Gene Disruptions

In these knockout experiments, the gene encoding PRO9852 polypeptides(designated as DNA129332-2775) (UNQ3037) was disrupted. The genespecific information for these studies is as follows: the mutated mousegene corresponds to nucleotide reference: NM_(—)145937 ACCESSION:NM_(—)145937 NID: gi 22122360 ref NM_(—)145937.1 Mus musculus ESTAI463102 (AI463102); protein reference: Q8ROF3 ACCESSION: Q8ROF3 NID:Mus musculus (Mouse). Sulfatase modifying factor 1 precursor(C-alpha-formlyglycine-generating enzyme 1); the human gene sequencereference: NM_(—)182760 Homo sapiens sulfatase modifying factor 1(SUMF1); the human protein sequence corresponds to reference: Q8NBK3ACCESSION: Q8NBK3 NID: Homo sapiens (Human). Sulfatase modifying factor1 precursor (C-alpha-formyglycine-generating enzyme 1).

The mouse gene of interest is Sumf1 (sulfatase modifying factor 1),ortholog of human SUMF1. Aliases include FGE, MGC39076, EST AI463102,and C-alpha-formylglycine-generating enzyme.

SUMF1 is an enzyme in the lumen of the endoplasmic reticulum thatcatalyzes the conversion of cysteine to C-alpha-formylglycine in thecatalytic site of various sulfatases, such as GALNS (galactosamine[N-acetyl]-6-sulfate sulfatase), ARSA (arylsulfatase A), STS (steroidsulfatase [microsomal], arylsulfatase C, isozyme S), and ARSE(arylsulfatase E [chondrodysplasia punctata 1]). This post-translationalmodification is required for enzymatic activity of these sulfatases.SUMF1 is expressed in a number of tissues, including heart, brain,placenta, lung, liver, skeletal muscle, kidney, and pancreas as well asin skin fibroblasts. Mutations in SUMF1 can cause multiple sulfatasedeficiency, a lysosomal storage disorder (OMIM 272200) (Cosma et al,Cell 113(4):421-2 (2003); Dierks et al, Cell 113(4):435-44 (2003); Cosmaet al, Hum Mutat 23(6):576-81 (2004); Preusser-Kunze et al, J Biol Chem280(15):14900-10 (2005)).

Targeted or gene trap mutations are generated in strain129SvEv^(Brd)-derived embryonic stem (ES) cells. The chimeric mice arebred to C57BL/6J albino mice to generate F1 heterozygous animals. Theseprogeny are intercrossed to generate F2 wild type, heterozygous, andhomozygous mutant progeny. On rare occasions, for example when very fewF1 mice are obtained from the chimera, F1 heterozygous mice are crossedto 129SvEv^(Brd)/C57 hybrid mice to yield additional heterozygousanimals for the intercross to generate the F2 mice. Level I phenotypicanalysis is performed on mice from this generation

wt het hom Total Observed 23 42 8 73 Expected 18.25 36.5 18.25 73Chi-Sq. = 13.92 Significance = 9.490965E−4 (hom/n) = 0.12 Avg. LitterSize = 9Mutation InformationMutation Type: Homologous Recombination (standard)Description: Coding exon 1 was targeted (NCBI accession NM_(—)145937.1).1. Wild-type Expression Panel: Expression of the target gene wasdetected in embryonic stem (ES) cells and in all 13 adult tissue samplestested by RT-PCR.2. QC Expression: QC Images: Disruption of the target gene was confirmedby Southern hybridization analysis.

48.44.1. Phenotypic Analysis (for Disrupted Gene: DNA129332-2775(UNQ3037)

(a) Overall Phenotypic Summary:

Mutation of the gene encoding the ortholog of human sulfatase modifyingfactor 1 (SUMF1) resulted in reduced viability and a lysosomal storagedisease in (−/−) mice. Genetic data indicate that this mutation resultedin reduced viability of the homozygous mutant mice. No homozygous mutantmice underwent complete Level 1 testing. The female mutant miceavailable for partial analysis exhibited signs of growth retardation andblood chemistry, immunological, and neurological abnormalities.Microscopic analysis revealed a lysosomal storage disease in themutants, characterized by macrophages distended by largeintracytoplasmic vesicles in all tissues. Disruption of the target genewas confirmed by Southern hybridization analysis.

(b) Pathology

Gross: The (−/−) mice exhibited several skeletal abnormalities,including convex sternum, vertebral kyphosis, shortened limbs.

Microscopic: The (−/−) mice exhibited a lysosomal storage disease,characterized by macrophages that were distended by largeintracytoplasmic vacuoles in all tissues. The affected macrophages weremost numerous in the red pulp of the spleen and lymph node sinuses, butdiffusely within the bones of the skull and the epiphyses of long bones.Kupffer cells in the liver and glial cells (primarily microglia) in thebrain and spinal cord were also distended by large cytoplasmic vacuoles.The large distended Kupffer cells bulged into and expanded the hepaticsinusoids. Some Ito cells also appeared to be hypertrophic. In thecentral nervous system, the affected microglial cells were most numerousin white tracts in the brain and spinal cord. Arterial (aortic) smoothmuscle cells were frequently distended by clear cytoplasmic vacuoles. Inthe more severely affected mutants, distended cells replaced the normalmarrow in the bones of the skull and surrounded and compressed thevestibulocochlear nerves. The lesions in long bones were most severe atthe metaphyses, where there was often complete absence of osteoblastsand trabecular bone, and increased numbers of osteoclasts. At theepiphyses, normal cellular elements were replaced by large distendedmacrophages, loose pale staining extracellular matrix, and scattereddetached chondrocytes. The normal maturation sequence of epiphysealcartilage was completely disrupted, characterized by an absence of thenormal columnar arrays of proliferating and hypertrophic chondrocytes.There was also reduced ossification of the epiphyseal cartilage templateof long bones and dysarthrosis. The grossly evident skeletalabnormalities reflect the defective bone development and maturation.Gene Expression: LacZ activity was not detected in the panel of tissuesby immunohistochemical analysis.

(c) Immunology Phenotypic Analysis

Immune related and inflammatory diseases are the manifestation orconsequence of fairly complex, often multiple interconnected biologicalpathways which in normal physiology are critical to respond to insult orinjury, initiate repair from insult or injury, and mount innate andacquired defense against foreign organisms. Disease or pathology occurswhen these normal physiological pathways cause additional insult orinjury either as directly related to the intensity of the response, as aconsequence of abnormal regulation or excessive stimulation, as areaction to self, or as a combination of these.

Though the genesis of these diseases often involves multistep pathwaysand often multiple different biological systems/pathways, interventionat critical points in one or more of these pathways can have anameliorative or therapeutic effect. Therapeutic intervention can occurby either antagonism of a detrimental process/pathway or stimulation ofa beneficial process/pathway.

T lymphocytes (T cells) are an important component of a mammalian immuneresponse. T cells recognize antigens which are associated with aself-molecule encoded by genes within the major histocompatibilitycomplex (MHC). The antigen may be displayed together with MHC moleculeson the surface of antigen presenting cells, virus infected cells, cancercells, grafts, etc. The T cell system eliminates these altered cellswhich pose a health threat to the host mammal. T cells include helper Tcells and cytotoxic T cells. Helper T cells proliferate extensivelyfollowing recognition of an antigen-MHC complex on an antigen presentingcell. Helper T cells also secrete a variety of cytokines, i.e.,lymphokines, which play a central role in the activation of B cells,cytotoxic T cells and a variety of other cells which participate in theimmune response.

In many immune responses, inflammatory cells infiltrate the site ofinjury or infection. The migrating cells may be neutrophilic,eosinophilic, monocytic or lymphocytic as can be determined byhistological examination of the affected tissues. Current Protocols inImmunology, ed. John E. Coligan, 1994, John Wiley & Sons, Inc.

Many immune related diseases are known and have been extensivelystudied. Such diseases include immune-mediated inflammatory diseases(such as rheumatoid arthritis, immune mediated renal disease,hepatobiliary diseases, inflammatory bowel disease (IBD), psoriasis, andasthma), non-immune-mediated inflammatory diseases, infectious diseases,immunodeficiency diseases, neoplasia, and graft rejection, etc. In thearea of immunology, targets were identified for the treatment ofinflammation and inflammatory disorders.

In the area of immunology, targets have been identified herein for thetreatment of inflammation and inflammatory disorders. Immune relateddiseases, in one instance, could be treated by suppressing the immuneresponse. Using neutralizing antibodies that inhibit molecules havingimmune stimulatory activity would be beneficial in the treatment ofimmune-mediated and inflammatory diseases. Molecules which inhibit theimmune response can be utilized (proteins directly or via the use ofantibody agonists) to inhibit the immune response and thus ameliorateimmune related disease.

The following tests were performed:

Hematology Analysis:

Test Description: Blood tests are carried out by Abbott's Cell-Dyn3500R, an automated hematology analyzer. Some of its features include afive-part WBC differential. ‘Patient’ reports can cover over 22parameters in all.

Results:

Hematology: The female (−/−) mice exhibited decreased hemoglobin,hematocrit, mean corpuscular volume, mean corpuscular volume and meancorpuscular hemoglobin when compared with those of their (+/+)littermates and the historical means. The female (−/−) mice alsoexhibited an increased red cell distribution width.

The (−/−) mice exhibited a hemoglobin level, hematocrit and a decreasein corpuscular volume when compared with their (+/+) littermates and thehistorical means.

These results are related to a phenotype associated with anemia. Thus,PRO9852 polypeptides, agonists thereof or the encoding gene for PRO9852polypeptides must be essential for normal red blood cell production andas such would be useful in the treatment of blood disorders associatedwith anemia or a low hematocrit.

Fluorescence-Activated Cell-Sorting (FACS) Analysis

Procedure:

FACS analysis of immune cell composition from peripheral blood wasperformed including CD4, CD8 and T cell receptor to evaluate Tlymphocytes, CD19 for B lymphocytes, CD45 as a leukocyte marker and panNK for natural killer cells. The FACS analysis was carried out on wildtype and homozygous mice and included cells derived from thymus, spleen,bone marrow and lymph node.

In these studies, analyzed cells were isolated from thymus, peripheralblood, spleen, bone marrow and lymph nodes. Flow cytometry was designedto determine the relative proportions of CD4 and CD8 positive T cells, Bcells, NK cells and monocytes in the mononuclear cell population. ABecton-Dickinson FACS Calibur 3-laser FACS machine was used to assessimmune status. For Phenotypic Assays and Screening, this machine recordsCD4+/CD8−, CD8+/CD4−, NK, B cell and monocyte numbers in addition to theCD4+/CD8+ ratio.

The mononuclear cell profile was derived by staining a single sample oflysed peripheral blood from each mouse with a panel of sixlineage-specific antibodies: CD45 PerCP, anti-TCRb APC, CD4 PE, CD8FITC, pan-NK PE, and CD19 FITC. The two FITC and PE labeled antibodiesstain mutually exclusive cell types. The samples were analyzed using aBecton Dickinson FACS Calibur flow cytometer with CellQuest software.

Results:

FACS3: The (−/−) mice exhibited an altered distribution of leukocytesubsets in the peripheral blood, characterized by a decreased meanpercentage of B cells and an increased mean percentage of CD8 cells whencompared with those of their (+/+) littermates and the historical means.

These results show that knockout (−/−) mice exhibit immunologicalabnormalities compared to their wild-type (+/+) littermates. Antagonists(inhibitors) of PRO9852 polypeptides would be expected to mimic thisphenotype. PRO9852 polypeptides or agonists thereof appear to act as anegative regulator of T cell production and a positive regulator of Bcell development and would be useful in the development or maturation ofB cells which could then participate in fast immune responses.

Ovalbumin Challenge

Procedure: This assay was carried out on wild type mice and homozygousmice. Chicken ovalbumin (OVA) is a T-cell dependent antigen, which iscommonly used as a model protein for studying antigen-specific immuneresponses in mice. OVA is non-toxic and inert and therefore will notcause harm to the animals even if no immune response is induced. Themurine immune response to OVA has been well characterized, to the extentthat the immunodominant peptides for eliciting T cell responses havebeen identified. Anti-OVA antibodies are detectable 8 to 10 days afterimmunization using enzyme-linked immunosorbent assay (ELIZA), anddetermination of different isotypes of antibodies gives furtherinformation on the complex processes that may lead to a deficientresponse in genetically engineered mice.

As noted above, this protocol assesses the ability of mice to raise anantigen-specific immune response. Animals were injected IP with 50 mg ofchicken ovalbumin emulsified in Complete Freund's Adjuvant and 14 dayslater the serum titer of anti-ovalbumin antibodies (IgM, IgG1 and IgG2subclasses) was measured. The amount of OVA-specific antibody in theserum sample is proportional to the Optical Density (OD) value generatedby an instrument that scans a 96-well sample plate. Data was collectedfor a set of serial dilutions of each serum sample.

Results of this Challenge:

The (−/−) mice exhibited decreased mean serum IgG1 and IgG2a responseswhen compared with their (+/+) littermates and the historical mean.

In summary, the ovalbumin challenge studies indicate that knockout micedeficient in the gene encoding PRO9852 polypeptides exhibitimmunological abnormalities when compared with their wild-typelittermates. In particular, the mutant mice exhibited a decreasedability to elicit an immunological response when challenged with theT-cell dependent OVA antigen. Thus, PRO9852 polypeptides or agoniststhereof, would be useful for stimulating the immune system (such as Tcell proliferation) and would find utility in the cases wherein thiseffect would be beneficial to the individual such as in the case ofleukemia, and other types of cancer, and in immunocompromised patients,such as AIDS sufferers. Accordingly, inhibitors (antagonists) of PRO9852polypeptides would be useful for inhibiting the immune response and thuswould be useful candidates for suppressing harmful immune responses,e.g. in the case of graft rejection or graft-versus-host diseases.

(d) Phenotypic Analysis: CNS/Neurology

In the area of neurology, analysis focused herein on identifying in vivovalidated targets for the treatment of neurological and psychiatricdisorders including depression, generalized anxiety disorders, attentiondeficit hyperactivity disorder, obsessive compulsive disorder,schizophrenia, cognitive disorders, hyperalgesia and sensory disorders.Neurological disorders include the category defined as “anxietydisorders” which include but are not limited to: mild to moderateanxiety, anxiety disorder due to a general medical condition, anxietydisorder not otherwise specified, generalized anxiety disorder, panicattack, panic disorder with agoraphobia, panic disorder withoutagoraphobia, posttraumatic stress disorder, social phobia, specificphobia, substance-induced anxiety disorder, acute alcohol withdrawal,obsessive compulsive disorder, agoraphobia, bipolar disorder I or II,bipolar disorder not otherwise specified, cyclothymic disorder,depressive disorder, major depressive disorder, mood disorder,substance-induced mood disorder. In addition, anxiety disorders mayapply to personality disorders including but not limited to thefollowing types: paranoid, antisocial, avoidant behavior, borderlinepersonality disorders, dependent, histronic, narcissistic,obsessive-compulsive, schizoid, and schizotypal.

Procedure:

Behavioral screens were performed on a cohort of wild type, heterozygousand homozygous mutant mice. All behavioral tests were done between 12and 16 weeks of age unless reduced viability necessitates earliertesting. These tests included open field to measure anxiety, activitylevels and exploration.

Open Field Test:

Several targets of known drugs have exhibited phenotypes in the openfield test. These include knockouts of the seratonin transporter, thedopamine transporter (Giros et al., Nature. 1996 Feb. 15;379(6566):606-12), and the GABA receptor (Homanics et al., Proc NatlAcad Sci USA. 1997 Apr. 15; 94(8):4143-8). An automated open-field assaywas customized to address changes related to affective state andexploratory patterns related to learning. First, the field (40×40 cm)was selected to be relatively large for a mouse, thus designed to pickup changes in locomotor activity associated with exploration. Inaddition, there were 4 holes in the floor to allow for nose-poking, anactivity specifically related to exploration. Several factors were alsodesigned to heighten the affective state associated with this test. Theopen-field test is the first experimental procedure in which the miceare tested, and the measurements that were taken were the subjects'first experience with the chamber. In addition, the open-field wasbrightly lit. All these factors will heighten the natural anxietyassociated with novel and open spaces. The pattern and extent ofexploratory activity, and especially the center-to-total distancetraveled ratio, may then be able to discern changes related tosusceptibility to anxiety or depression. A large arena (40 cm×40 cm,VersaMax animal activity monitoring system from AccuScan Instruments)with infrared beams at three different levels was used to recordrearing, hole poke, and locomotor activity. The animal was placed in thecenter and its activity was measured for 20 minutes. Data from this testwas analyzed in five, 4-minute intervals. The total distance traveled(cm), vertical movement number (rearing), number of hole pokes, and thecenter to total distance ratio were recorded.

The propensity for mice to exhibit normal habituation responses to anovel environment is assessed by determining the overall change in theirhorizontal locomotor activity across the 5 time intervals. Thiscalculated slope of the change in activity over time is determined usingnormalized, rather than absolute, total distance traveled. The slope isdetermined from the regression line through the normalized activity ateach of the 5 time intervals. Normal habituation is represented by anegative slope value.

Results:

The (−/−) mice exhibited an increased median sum time and distancein-center during open field testing when compared with theirgender-matched (+/+) littermates and the historical mean, suggesting adecreased anxiety-like response in the mutants.

A notable difference was observed during open field activity testing.The (−/−) mice exhibited an increased median sum time in the center areawhen compared with their gender-matched (+/+) littermates, which isindicative of a decreased anxiety-like response in the mutants. Thus,knockout mice demonstrated a phenotype consistent with depression,generalized anxiety disorders, cognitive disorders, hyperalgesia andsensory disorders and/or bipolar disorders. Thus, PRO9852 polypeptidesand agonists thereof would be useful for the treatment or ameliorationof the symptoms associated with depressive disorders.

Functional Observational Battery (FOB) Test—Tail Suspension Testing:

The FOB is a series of situations applied to the animal to determinegross sensory and motor deficits. A subset of tests from the Irwinneurological screen that evaluates gross neurological function is used.In general, short-duration, tactile, olfactory, and visual stimuli areapplied to the animal to determine their ability to detect and respondnormally. These simple tests take approximately 10 minutes and the mouseis returned to its home cage at the end of testing.

Tail Suspension Testing:

The tail suspension test is a procedure that has been developed as amodel for depressive-like behavior in rodents. In this particular setup,a mouse is suspended by its tail for 6 minutes, and in response themouse will struggle to escape from this position. After a certain periodof time the struggling of the mouse decreases and this is interpreted asa type of learned helplessness paradigm. Animals with invalid data (i.e.climbed their tail during the testing period) are excluded fromanalysis.

Results:

Tail Suspension2: The female (−/−) mice exhibited increased immobilitytime when compared with that of their gender-matched (+/+) littermatesand the historical mean, suggesting an increased depressive-likeresponse in the mutants.

Thus, knockout mice demonstrated a phenotype consistent with depression,generalized anxiety disorders, cognitive disorders, hyperalgesia andsensory disorders and/or bipolar disorders. Thus, PRO9852 polypeptidesand agonists thereof would be useful for the treatment or ameliorationof the symptoms associated with depressive disorders.

Prepulse Inhibition of the Acoustic Startle Reflex

Prepulse inhibition of the acoustic startle reflex occurs when a loud120 decibel (dB) startle-inducing tone is preceded by a softer(prepulse) tone. The PPI paradigm consists of six different trial types(70 dB background noise, 120 dB alone, 74 dB+120 dB−pp4, 78 dB+120dB−pp8, 82 dB+120 dB−pp12, and 90 dB+120 dB−pp20) each repeated inpseudo random order six times for a total of 36 trials. The max responseto the stimulus (V max) is averaged for each trial type. Animals with a120 dB average value equal to or below 100 are excluded from analysis.The percent that the prepulse inhibits the animal's response to thestartle stimulus is calculated and graphed.

Results:

PPI: The (−/−) mice failed to exhibit a startle response, suggestinghearing impairment in the mutants.

Therefore, prepulse inhibition could not be assessed.

Circadian Test Description:

Female mice are individually housed at 4 pm on the first day of testingin 48.2 cm×26.5 cm home cages and administered food and water adlibitum. Animals are exposed to a 12-hour light/dark cycle with lightsturning on at 7 am and turning off at 7 pm. The system software recordsthe number of beam interruptions caused by the animal's movements, withbeam breaks automatically divided into ambulations. Activity is recordedin 60, one-hour intervals during the three-day test. Data generated aredisplayed by median activity levels recorded for each hour (circadianrhythm) and median total activity during each light/dark cycle(locomotor activity) over the three-day testing period.

Results:

Circadian: The female (−/−) mice exhibited decreased ambulatory countsduring the 1- and 12-hour habituation periods and all light/dark periodswhen compared with their gender-matched (+/+) littermates and thehistorical means.

These results are consistent with lethargy or depressive disorders.Antagonists or inhibitors of PRO9852 polypeptides or the PRO9852encoding gene would be expected to mimic this behavior. Likewise,PRO9852 polypeptides or agonists thereof, would be useful in thetreatment of such neurological disorders including depressive disordersor other decreased anxiety-like symptoms such as lethargy, cognitivedisorders, hyperalgesia and sensory disorders.

Inverted Screen Testing:

Behavioral screens were performed on a cohort of wild type, heterozygousand homozygous mutant mice. All behavioral tests were done between 12and 16 weeks of age unless reduced viability necessitates earliertesting. These tests included open field to measure anxiety, activitylevels and exploration.

Inverted Screen Test Data:

The Inverted Screen is used to measure motor strength/coordination.Untrained mice were placed individually on top of a square (7.5 cm×7.5cm) wire screen which was mounted horizontally on a metal rod. The rodwas then rotated 180 degrees so that the mice were on the bottom of thescreens. The following behavioral responses were recorded over a 1 mintesting session: fell off, did not climb, and climbed up.

Results:

Genotype Ratio Fell Down % Ratio Climbed up % +/+ (n = 8) 1/8 13 7/887.5 −/− (n = 8) 5/8 63 0/8 0WT Population Fell Down 3.62 Climbed Up 60.04A motor strength deficit is apparent when there is a 50% pointdifference between (−/−) or (+/−) mice and (+/+) mice for the fell downresponse. 0/8 or 1/8 (−/−) or (+/−) mice not climbing indicates impairedmotor coordination. 7/8 or 8/8(−/−) or (+/−) mice climbing up indicatesenhanced motor coordination.

The Inverted Screen Test is designed to measure basic sensory & motorobservations:

Among the 8 (−/−) mice analyzed, 5 fell off the inverted screen whereasonly 1/8 (+/+) mice fell off. These results indicate an impaired motorstrength in the mutants. These results are consistent with theobservations in bone-related measurements as shown below.

(e) Phenotypic Analysis: Metabolism—Blood Chemistry

In the area of metabolism, targets may be identified for the treatmentof diabetes. Blood chemistry phenotypic analysis includes blood glucosemeasurements. The COBAS Integra 400 (mfr: Roche) was used for runningblood chemistry tests on the mice. In addition to measuring bloodglucose levels the following blood chemistry tests are also routinelyperformed: Alkaline Phosphatase; Alanine Amino-Transferase; Albumin;Bilirubin; Phosphorous; Creatinine; BUN=Blood Urea Nitrogen; Calcium;Uric Acid; Sodium; Potassium; and Chloride. In the area of metabolism,targets may be identified for the treatment of diabetes. Blood chemistryphenotypic analysis includes glucose tolerance tests to measure insulinsensitivity and changes in glucose metabolism. Abnormal glucosetolerance test results may indicate but may not be limited to thefollowing disorders or conditions: Diabetes Type 1 and Type 2, SyndromeX, various cardiovascular diseases and/or obesity.

Results:

Blood Chemistry: The female (−/−) mice exhibited increased alkalinephosphatase, albumin, alanine amino transferase, phosphorus, andpotassium levels when compared with those of their (+/+) littermates andthe historical means. These blood chemistry abnormalities are consistentwith the reduced viability consequences when the PRO9852 encoding geneis knocked out in mice.

(f) Bone Metabolism & Body Diagnostics

(1) Tissue Mass & Lean Body Mass Measurements—Dexa

Dexa Analysis—Test Description:

Procedure: A cohort of wild type, heterozygous and homozygous mice weretested in this assay. Dual Energy X-ray Absorptiometry (DEXA) has beenused successfully to identify changes in total tissue mass (TTM).

The mouse was anesthetized by intraperitoneal injection of Avertin(1.25% 2,2,2,-tribromoethanol, 20 ml/kg body weight), body length andweight were measured, and then the mouse was placed in a prone positionon the platform of the PIXImus™ Densitometer (Lunar Inc.) for a DEXAscan. Using Lunar PIXImus software, the bone mineral density (BMD) andfat composition (% fat) and total tissue mass (TTM) were determined inthe regions of interest (ROI, i.e., whole body, vertebrae, and bothfemurs).

Body Measurements (Body Length & Weight):

Body Measurements: A measurement of body length and weight was performedat approximately 16 weeks of age.

Results:

Weight: The (−/−) mice exhibited decreased mean body weight whencompared with that of their gender-matched (+/+) littermates and thehistorical mean.

Length: The 2 female (−/−) mice analyzed exhibited decreased mean bodylength when compared with that of their gender-matched (+/+) littermatesand the historical mean.

(2) Bone Metabolism: Radiology Phenotypic Analysis

In the area of bone metabolism, targets were identified herein for thetreatment of arthritis, osteoporosis, osteopenia and osteopetrosis aswell as identifying targets that promote bone healing. Tests included:

DEXA for measurement of bone mineral density on femur and vertebra

MicroCT for very high resolution and very high sensitivity measurementsof bone mineral density for both trabecular and cortical bone.

Dexa Analysis—Test Description:

Procedure: A cohort of wild type, heterozygous and homozygous mice weretested in this assay. Dual Energy X-ray Absorptiometry (DEXA) has beenused successfully to identify changes in bone. Anesthetized animals wereexamined and bone mineral content (BMC), BMC/LBM ratios, volumetric bonemineral density (vBMD), total body BMD, femur BMD and vertebra BMD weremeasured.

The mouse was anesthetized by intraperitoneal injection of Avertin(1.25% 2,2,2,-tribromoethanol, 20 ml/kg body weight), body length andweight were measured, and then the mouse was placed in a prone positionon the platform of the PIXImus™ Densitometer (Lunar Inc.) for a DEXAscan. Using Lunar PIXImus software, the bone mineral density (BMD) andfat composition (% fat) and total tissue mass (TTM) were determined inthe regions of interest (ROI) [i.e., whole body, vertebrae, and bothfemurs].

Results:

DEXA: The 2 female (−/−) mice available for analysis exhibited decreasedtotal tissue mass, lean body mass, percent total body fat, total fatmass, and total body bone mineral content when compared with thehistorical means. However, the 2 female (+/+) mice also exhibitedsimilarly decreased measurements.Micro CT: No notable difference. However, no (−/−) mice were availablefor analysis.

Mutant (−/−) mice deficient in the gene encoding PRO9852 polypeptidesshow a phenotype consistent with growth retardation, marked by decreasedbody weight and length and tissue wasting diseases (decreased total bodyfat (%) and fat mass (g)). Thus, antagonists or inhibitors of PRO9852polypeptides or its encoding gene would mimic these metabolic and growthrelated effects. On the other hand, PRO9852 polypeptides or agoniststhereof would be useful in the prevention and/or treatment of suchmetabolic disorders as diabetes or other tissue wasting diseases.

In addition, the (−/−) mice analyzed by DEXA exhibited decreased bonemeasurements and decreased body mass measurements when compared withtheir (+/+) littermates, suggestive of abnormal bone disorders. Inaddition, the decreased mean total tissue mass and lean body mass isindicative of a metabolic disorder related to growth retardation andtissue wasting disorders. The negative bone phenotype indicates thatPRO9852 polypeptides or agonists thereof would be useful for maintainingbone homeostasis in addition to normal growth development. In addition,PRO9852 polypeptides would be useful in bone healing or for thetreatment of arthritis or osteoporosis, whereas antagonists (orinhibitors) of PRO9852 polypeptides or its encoding gene would lead toabnormal or pathological bone disorders including inflammatory diseasesassociated with abnormal bone metabolism including arthritis,osteoporosis and osteopenia.

These findings are consistent with the pathology report and themicroscopic observations.

(g) Cardiology/Diagnostics—Blood Pressure

Description:

Systolic blood pressure is measured via a noninvasive tail-cuff methodfor four days on the Visitech BP-2000 Blood Pressure Analysis System.The blood pressure is measured ten times each day for four days. Thefour days are then averaged to obtain a mouse's conscious systolic bloodpressure.

Results:

Blood Pressure: The 2 female (−/−) mice available for analysis exhibitedincreased mean systolic blood pressure when compared with that of theirgender-matched (+/+) littermates and the historical mean which isindicative of hypertension.

48.45. Generation and Analysis of Mice Comprising DNA143076-2787(UNQ3054) Gene Disruptions

In these knockout experiments, the gene encoding PRO9873 polypeptides(designated as DNA143076-2787) (UNQ3054) was disrupted. The genespecific information for these studies is as follows: the mutated mousegene corresponds to nucleotide reference: NM_(—)020595 Mus musculusotoraplin (Otor); protein reference: Q9JIE3 ACCESSION: Q9JIE3 NID: Musmusculus (Mouse). Otoraplin precursor (Melanoma inhibitory activity-likeprotein); the human gene sequence reference: NM_(—)020157 ACCESSION:NM_(—)020157 NID: gi 21618345 ref NM_(—)020157.2 Homo sapiens otoraplin(OTOR); the human protein sequence corresponds to reference: Q9NRC9ACCESSION: Q9NRC9 NID: Homo sapiens (Human). Otoraplin precursor(Fibrocyte-derived protein) (Melanoma inhibitory activity like protein).

The mouse gene of interest is Otor (otoraplin), ortholog of human OTOR.Aliases include Fdp, MIA, MIAL, CDRAP, fibrocyte-derived protein, andmelanoma inhibitory activity-like protein.

OTOR is a secreted protein expressed primarily by the mesenchymal celllayer beneath sensory epithelium of the cochlea and vestibule of theinner ear. The protein consists of a signal peptide and an SH3 domainand undergoes post-translational modification, resulting in sulfationand covalent homodimerization (Rendtorff et al, Genomics 71(1):40-52(2001); Stoll et al, Protein Sci 12(3):510-9 (2003); Robertson et al,Genomics 66(3):242-8 (2000)). OTOR may function as a component ofextracellular matrix or as a signal-transducing ligand (Bosserhoff andBuettner, Histol Histonathol 17(1):289-300 (2002)). OTOR likely plays arole in periotic mesenchyme chondrogenesis, participating in formationof the otic capsule during development (Cohen-Salmon et al, J Biol Chem275(51):40036-41 (2000)). Mutations in OTOR may cause deafness(Cohen-Salmon et al, J Biol Chem 275(51):40036-41 (2000); Rendtorff etal, Genomics 71(1):40-52 (2001).

Targeted or gene trap mutations are generated in strain129SvEv^(Brd)-derived embryonic stem (ES) cells. The chimeric mice arebred to C57BL/6J albino mice to generate F1 heterozygous animals. Theseprogeny are intercrossed to generate F2 wild type, heterozygous, andhomozygous mutant progeny. On rare occasions, for example when very fewF1 mice are obtained from the chimera, F1 heterozygous mice are crossedto 129SvEv^(Brd)/C57 hybrid mice to yield additional heterozygousanimals for the intercross to generate the F2 mice. Level I phenotypicanalysis is performed on mice from this generation

wt het hom Total Observed 20 30 29 79 Expected 19.75 39.5 19.75 79Chi-Sq. = 0.81 Significance = 0.6669768 (hom/n) = 0.27 Avg. Litter Size= 9Mutation InformationMutation Type: Homologous Recombination (standard)Description: Coding exons 1 through 3 were targeted (NCBI accessionNM_(—)020595.1).1. Wild-type Expression Panel: Expression of the target gene wasdetected in brain, spinal cord, eye, and thymus among 13 adult tissuesamples tested by RT-PCR.2. QC Expression: QC Images: Disruption of the target gene was confirmedby Southern hybridization analysis.

48.45.1. Phenotypic Analysis (for Disrupted Gene: DNA143076-2787(UNQ3054)

(a) Overall Phenotypic Summary:

Mutation of the gene encoding the ortholog of human otoraplin (OTOR)resulted in an increased anxiety-related response in male (−/−) mice.Gene disruption was confirmed by Southern blot.

(b) Phenotypic Analysis: CNS/Neurology

In the area of neurology, analysis focused herein on identifying in vivovalidated targets for the treatment of neurological and psychiatricdisorders including depression, generalized anxiety disorders, attentiondeficit hyperactivity disorder, obsessive compulsive disorder,schizophrenia, cognitive disorders, hyperalgesia and sensory disorders.Neurological disorders include the category defined as “anxietydisorders” which include but are not limited to: mild to moderateanxiety, anxiety disorder due to a general medical condition, anxietydisorder not otherwise specified, generalized anxiety disorder, panicattack, panic disorder with agoraphobia, panic disorder withoutagoraphobia, posttraumatic stress disorder, social phobia, specificphobia, substance-induced anxiety disorder, acute alcohol withdrawal,obsessive compulsive disorder, agoraphobia, bipolar disorder I or II,bipolar disorder not otherwise specified, cyclothymic disorder,depressive disorder, major depressive disorder, mood disorder,substance-induced mood disorder. In addition, anxiety disorders mayapply to personality disorders including but not limited to thefollowing types: paranoid, antisocial, avoidant behavior, borderlinepersonality disorders, dependent, histronic, narcissistic,obsessive-compulsive, schizoid, and schizotypal.

Procedure:

Behavioral screens were performed on a cohort of 4 wild type, 4heterozygous and 8 homozygous mutant mice. All behavioral tests weredone between 12 and 16 weeks of age unless reduced viabilitynecessitates earlier testing. These tests included open field to measureanxiety, activity levels and exploration.

Open Field Test:

Several targets of known drugs have exhibited phenotypes in the openfield test. These include knockouts of the seratonin transporter, thedopamine transporter (Giros et al., Nature. 1996 Feb. 15;379(6566):606-12), and the GABA receptor (Homanics et al., Proc NatlAcad Sci USA. 1997 Apr. 15; 94(8):4143-8). An automated open-field assaywas customized to address changes related to affective state andexploratory patterns related to learning. First, the field (40×40 cm)was selected to be relatively large for a mouse, thus designed to pickup changes in locomotor activity associated with exploration. Inaddition, there were 4 holes in the floor to allow for nose-poking, anactivity specifically related to exploration. Several factors were alsodesigned to heighten the affective state associated with this test. Theopen-field test is the first experimental procedure in which the miceare tested, and the measurements that were taken were the subjects'first experience with the chamber. In addition, the open-field wasbrightly lit. All these factors will heighten the natural anxietyassociated with novel and open spaces. The pattern and extent ofexploratory activity, and especially the center-to-total distancetraveled ratio, may then be able to discern changes related tosusceptibility to anxiety or depression. A large arena (40 cm×40 cm,VersaMax animal activity monitoring system from AccuScan Instruments)with infrared beams at three different levels was used to recordrearing, hole poke, and locomotor activity. The animal was placed in thecenter and its activity was measured for 20 minutes. Data from this testwas analyzed in five, 4-minute intervals. The total distance traveled(cm), vertical movement number (rearing), number of hole pokes, and thecenter to total distance ratio were recorded.

The propensity for mice to exhibit normal habituation responses to anovel environment is assessed by determining the overall change in theirhorizontal locomotor activity across the 5 time intervals. Thiscalculated slope of the change in activity over time is determined usingnormalized, rather than absolute, total distance traveled. The slope isdetermined from the regression line through the normalized activity ateach of the 5 time intervals. Normal habituation is represented by anegative slope value.

Results:

Anxiety: The male (−/−) mice exhibited decreased median sumtime-in-center during open field testing when compared with theirgender-matched (+/+) littermates and the historical mean, suggesting anincreased anxiety-like response in the mutants.

The (−/−) mice demonstrated a decrease median sum time-in-center atintervals 2,3, and 5 when compared to the (+/+) mice, suggesting anincreased anxiety-like response in the (−/−) mice. In summary, the openfield testing revealed a phenotype associated with increased anxietywhich could be associated with mild to moderate anxiety, anxiety due toa general medical condition, and/or bipolar disorders; hyperactivity;sensory disorders; obsessive-compulsive disorders, schizophrenia or aparanoid personality. Thus, PRO9873 polypeptides or agonists thereofwould be useful in the treatment of such neurological disorders.

48.46. Generation and Analysis of Mice Comprising DNA144841-2816(UNQ3115) Gene Disruptions

In these knockout experiments, the gene encoding PRO10196 polypeptides(designated as DNA144841-2816) (UNQ3115) was disrupted. The genespecific information for these studies is as follows: the mutated mousegene corresponds to nucleotide reference: NM_(—)020013 Mus musculusfibroblast growth factor 21 (Fgf21); protein reference: Q9JJN1ACCESSION: Q9JJN1 NID: Mus musculus (Mouse). Fibroblast growth factor-21precursor (FGF-21); the human gene sequence reference: NM_(—)019113ACCESSION: NM_(—)019113 NID:9506596 Homo sapiens fibroblast growthfactor 21 (FGF21); the human protein sequence corresponds to reference:Q9NSA1 ACCESSION: Q9NSA1 NID: Homo sapiens (Human). FIBROBLAST GROWTHFACTOR-21 PRECURSOR (FGF-21).

The mouse gene of interest is Fgf21 (fibroblast growth factor 21),ortholog of human FGF21. Aliases include FGF-21 and UNQ3115.

FGF21 is a putative secreted protein expressed primarily in liver. The209-amino acid protein contains a signal peptide and a fibroblast growthfactor (FGF) domain (Pfam accession PF00167). FGF21 may function as asignal-transducing ligand (Nishimura et al, Biochim Biophys Acta1492(1):203-6 (2000)).

Targeted or gene trap mutations are generated in strain129SvEv^(Brd)-derived embryonic stem (ES) cells. The chimeric mice arebred to C57BL/6J albino mice to generate F1 heterozygous animals. Theseprogeny are intercrossed to generate F2 wild type, heterozygous, andhomozygous mutant progeny. On rare occasions, for example when very fewF1 mice are obtained from the chimera, F1 heterozygous mice are crossedto 129SvEv^(Brd)/C57 hybrid mice to yield additional heterozygousanimals for the intercross to generate the F2 mice. Level I phenotypicanalysis is performed on mice from this generation

wt het hom Total Observed 17 49 21 87 Expected 21.75 43.5 21.75 87Chi-Sq. = 2.45 Significance = 0.2937577 (hom/n) = 0.25 Avg. Litter Size= 9Mutation InformationMutation Type Homologous Recombination (standard)Description: Coding exons 1 through 3 were targeted (NCBI accessionNM_(—)020013.2).1. Wild-type Expression Panel: Expression of the target gene wasdetected only in brain among the 13 adult tissue samples tested byRT-PCR.2. QC Expression: QC Images: Disruption of the target gene was confirmedby Southern hybridization analysis.

48.46.1. Phenotypic Analysis (for Disrupted Gene: DNA144841-2816(UNQ3115)

(a) Overall Phenotypic Summary:

Mutation of the gene encoding the ortholog of human fibroblast growthfactor 21 (FGF21) resulted in the (−/−) mice exhibiting increased meanserum cholesterol and glucose levels. The mutant (−/−) mice also showedincreased total tissue mass and total body fat. Gene disruption wasconfirmed by Southern blot.

(b) Phenotypic Analysis: Cardiology

In the area of cardiovascular biology, targets were identified hereinfor the treatment of hypertension, atherosclerosis, heart failure,stroke, various coronary artery diseases, dyslipidemias such as highcholesterol (hypercholesterolemia) and elevated serum triglycerides(hypertriglyceridemia), diabetes and/or obesity. The phenotypic testsincluded the measurement of serum cholesterol and triglycerides.

Blood Lipids

Procedure: A cohort of 4 wild type, 4 heterozygous and 8 homozygous micewere tested in this assay. High cholesterol levels and increasedtriglyceride blood levels are recognized risk factors in the developmentof cardiovascular disease and/or diabetes. Measuring blood lipidsfacilitates the finding of biological switches that regulate blood lipidlevels. Inhibition of factors which elevate blood lipid levels may beuseful for reducing the risk for cardiovascular disease. In these bloodchemistry tests, measurements were recorded using the COBAS Integra 400(mfr: Roche).

Results:

Blood Chemistry: The (−/−) mice exhibited an increased mean serumcholesterol level when compared with that of their gender-matched (+/+)littermates and the historical mean.

As summarized above, the (−/−) mice exhibited increased mean serumcholesterol levels when compared with their gender-matched (+/+)littermates and the historical means. Thus, mutant mice deficient in thePRO10196 gene may serve as a model for cardiovascular disease. PRO10196polypeptides or its encoding gene would be useful in regulating bloodlipids such as cholesterol. Thus, PRO10196 polypeptides or agoniststhereof would be useful in the treatment of such cardiovascular diseasesas hypertension, atherosclerosis, heart failure, stroke, variouscoronary diseases, hypercholesterolemia, diabetes and/or obesity.

(c) Phenotypic Analysis: Metabolism—Blood Chemistry

In the area of metabolism, targets may be identified for the treatmentof diabetes. Blood chemistry phenotypic analysis includes blood glucosemeasurements. The COBAS Integra 400 (mfr: Roche) was used for runningblood chemistry tests on the mice. In the area of metabolism, targetsmay be identified for the treatment of diabetes.

Results:

The homozygous (−/−) mice exhibited increased mean serum glucose levelswhen compared with that of their gender-matched (+/+) littermates andthe historical mean.

Thus, the mutant (−/−) mice exhibited hyperglycemia which could beassociated with an altered glucose metabolism or diabetes. PRO10196polypeptides or agonists thereof would be useful in maintaining normalglucose levels/metabolism and possibly useful in the treatment ofdiabetes.

(d) Bone Metabolism & Body Diagnostics: Radiology Phenotypic Analysis

In the area of bone metabolism, targets were identified herein for thetreatment of arthritis, osteoporosis, osteopenia and osteopetrosis aswell as identifying targets that promote bone healing. Tests included:

DEXA for measurement of bone mineral density on femur and vertebra

MicroCT for very high resolution and very high sensitivity measurementsof bone mineral density for both trabecular and cortical bone.

Dexa Analysis—Test Description:

Procedure: A cohort of 4 wild type, 4 heterozygous and 8 homozygous micewere tested in this assay. Dual Energy X-ray Absorptiometry (DEXA) hasbeen used successfully to identify changes in bone. Anesthetized animalswere examined and bone mineral content (BMC), BMC/LBM ratios, volumetricbone mineral density (vBMD), total body BMD, femur BMD and vertebra BMDwere measured.

The mouse was anesthetized by intraperitoneal injection of Avertin(1.25% 2,2,2,-tribromoethanol, 20 ml/kg body weight), body length andweight were measured, and then the mouse was placed in a prone positionon the platform of the PIXImus™ Densitometer (Lunar Inc.) for a DEXAscan. Using Lunar PIXImus software, the bone mineral density (BMD) andfat composition (% fat) and total tissue mass (TTM) were determined inthe regions of interest (ROI) [i.e., whole body, vertebrae, and bothfemurs].

Results:

The male (−/−) mice exhibited increased total tissue mass and total bodyfat (% and g) when compared to their gender matched wild-type (+/+)littermates and historical mean.

These studies suggest that mutant (−/−) non-human transgenic animalsexhibit a negative phenotype that would be associated with obesity.Thus, PRO10196 polypeptides or agonists thereof are essential for normalgrowth and metabolic processes and especially would be important in theprevention and/or treatment of obesity.

48.47. Generation and Analysis of Mice Comprising DNA220432 (UNQ3966)Gene Disruptions

In these knockout experiments, the gene encoding PRO34778 polypeptides(designated as DNA220432) (UNQ3966) was disrupted. The gene specificinformation for these studies is as follows: the mutated mouse genecorresponds to nucleotide reference: NM_(—)130866 Mus musculus olfactoryreceptor 78 (Olfr78); protein reference: Q8VBV9 Q8VBV9 Q8VBV9 OLFACTORYRECEPTOR MOR18-2 PROSTATE-SPECIF; the human gene sequence reference:NM_(—)030774 ACCESSION: NM_(—)030774 NID:19923630 Homo sapiens olfactoryreceptor, family 51, subfamily E, member 2 (ORS1E2); the human proteinsequence corresponds to reference: Q9H255 OXE2_HUMAN Q9H255 OLFACTORYRECEPTOR 51E2 PROSTATE SPECI.

The mouse gene of interest is Olfr78 (olfactory receptor 78), orthologof human OR51E2 (olfactory receptor, family 51, subfamily E, member 2).Aliases include PSGR; RA1c; MOL2.3; MOR18-2; 4633402A21Rik; olfactoryreceptor MOR18-2; GA_x6K02T2PBJ9-5459657-5458695; OR52A2; OR51E3P;olfactory receptor OR11-16; prostate specific G-protein coupledreceptor; olfactory receptor, family 52, subfamily A, member 2;olfactory receptor, family 51, subfamily E, member 3 pseudogene.

OR51E2 is an integral membrane protein expressed primarily in prostategland that likely functions as a G protein-coupled receptor. The proteincontains a seven-transmembrane receptor (rhodopsin family) domain (PFAMaccession PF00001), displays marked similarity with olfactory receptorfamily members, and interacts with GNA12 (guanine nucleotide bindingprotein [G protein] alpha 12). OR511E2 is also expressed in olfactorytissue and the medulla oblongata of the brain in humans, in brain andcolon in mice, and in brain and liver in rats (Xu et al, Cancer Res60(23):6568-72 (2000); Yuan et al, Gene 278(1-2):41-51 (2001); Xia etal, Oncogene 20(41):5903-7 (2001)). Expression of OR51E2 is frequentlyupregulated in prostate cancer, suggesting that the protein may beuseful for early detection and treatment of prostate cancer (Weng et al,Int J Cancer 113(5):811-8 (2005)).

Targeted or gene trap mutations are generated in strain129SvEv^(Brd)-derived embryonic stem (ES) cells. The chimeric mice arebred to C57BL/6J albino mice to generate F1 heterozygous animals. Theseprogeny are intercrossed to generate F2 wild type, heterozygous, andhomozygous mutant progeny. On rare occasions, for example when very fewF1 mice are obtained from the chimera, F1 heterozygous mice are crossedto 129SvEv^(Brd)/C57 hybrid mice to yield additional heterozygousanimals for the intercross to generate the F2 mice. Level I phenotypicanalysis is performed on mice from this generation

wt het hom Total Observed 18 25 18 61 Expected 15.25 30.5 15.25 61Chi-Sq. = 0.32 Significance = 0.85214376 (hom/n) = 0.27 Avg. Litter Size= 8Mutation InformationMutation Type: Homologous Recombination (standard)Description: Coding exon 1 was targeted (NCBI accession NM_(—)130866.2).1. Wild-type Expression Panel: Expression of the target gene wasdetected in embryonic stem (ES) cells and in all 26 adult tissue samplestested by RT-PCR, except liver and adipose.2. QC Expression: QC Images: Disruption of the target gene was confirmedby Southern hybridization analysis.

48.47.1. Phenotypic Analysis (for Disrupted Gene: DNA220432 (UNQ3966)

(a) Overall Phenotypic Summary:

Mutation of the gene encoding the ortholog of human olfactory receptor,family 51, subfamily E, member 2 (OR51E2) resulted in the (−/−) miceexhibiting increased total tissue mass and total body fat as well asincreased cholesterol levels. An enhanced glucose tolerance was alsoobserved in the mutant (−/−) mice. Neurological testing showed increasedstress induced hyperthermia in the homozygous mice. Gene disruption wasconfirmed by Southern blot.

(b) Phenotypic Analysis: Cardiology

In the area of cardiovascular biology, targets were identified hereinfor the treatment of hypertension, atherosclerosis, heart failure,stroke, various coronary artery diseases, dyslipidemias such as highcholesterol (hypercholesterolemia) and elevated serum triglycerides(hypertriglyceridemia), diabetes and/or obesity. The phenotypic testsincluded the measurement of serum cholesterol and triglycerides.

Blood Lipids

Procedure: A cohort of 4 wild type, 4 heterozygous and 8 homozygous micewere tested in this assay. High cholesterol levels and increasedtriglyceride blood levels are recognized risk factors in the developmentof cardiovascular disease and/or diabetes. Measuring blood lipidsfacilitates the finding of biological switches that regulate blood lipidlevels. Inhibition of factors which elevate blood lipid levels may beuseful for reducing the risk for cardiovascular disease. In these bloodchemistry tests, measurements were recorded using the COBAS Integra 400(mfr: Roche).

Results:

Blood Chemistry: The (−/−) mice exhibited an increased mean serumcholesterol level when compared with that of their gender-matched (+/+)littermates and the historical mean.

As summarized above, the (−/−) mice exhibited increased mean serumcholesterol levels when compared with their gender-matched (+/+)littermates and the historical means. Thus, mutant mice deficient in thePRO34778 gene may serve as a model for cardiovascular disease. PRO34778polypeptides or its encoding gene would be useful in regulating bloodlipids such as cholesterol. Thus, PRO34778 polypeptides or agoniststhereof would be useful in the treatment of such cardiovascular diseasesas hypertension, atherosclerosis, heart failure, stroke, variouscoronary diseases, hypercholesterolemia, diabetes and/or obesity.

(c) Phenotypic Analysis: Metabolism—Blood Chemistry/Glucose Tolerance

In the area of metabolism, targets may be identified for the treatmentof diabetes. Blood chemistry phenotypic analysis includes blood glucosemeasurements. The COBAS Integra 400 (mfr: Roche) was used for runningblood chemistry tests on the mice. In the area of metabolism, targetsmay be identified for the treatment of diabetes. Blood chemistryphenotypic analysis includes glucose tolerance tests to measure insulinsensitivity and changes in glucose metabolism. Abnormal glucosetolerance test results may indicate but may not be limited to thefollowing disorders or conditions: Diabetes Type 1 and Type 2, SyndromeX, various cardiovascular diseases and/or obesity.

Procedure: A cohort of 2 wild type and 4 homozygous mice were used inthis assay. The glucose tolerance test is the standard for definingimpaired glucose homeostasis in mammals. Glucose tolerance tests wereperformed using a Lifescan glucometer. Animals were injected IP at 2g/kg with D-glucose delivered as a 20% solution and blood glucose levelswere measured at 0, 30, 60 and 90 minutes after injection.

Results:

Glucose Tolerance Test: The mutant (−/−) mice tested exhibited enhancedglucose tolerance when compared with their gender-matched (+/+)littermates.

In these studies the mutant (−/−) mice showed an increased or enhancedglucose tolerance in the presence of normal fasting glucose at all 3intervals tested when compared with their gender-matched (+/+)littermates and the historical means. Thus, knockout mice exhibited anincreased insulin sensitivity or the opposite phenotypic pattern of animpaired glucose homeostasis, and as such antagonists (inhibitors) toPRO34778 polypeptides or its encoding gene would be useful in thetreatment of an impaired glucose homeostasis.

(d) Bone Metabolism & Body Diagnostics: Radiology Phenotypic Analysis

In the area of bone metabolism, targets were identified herein for thetreatment of arthritis, osteoporosis, osteopenia and osteopetrosis aswell as identifying targets that promote bone healing. Tests included:

DEXA for measurement of bone mineral density on femur and vertebra

MicroCT for very high resolution and very high sensitivity measurementsof bone mineral density for both trabecular and cortical bone.

Dexa Analysis—Test Description:

Procedure: A cohort of 4 wild type, 4 heterozygous and 8 homozygous micewere tested in this assay. Dual Energy X-ray Absorptiometry (DEXA) hasbeen used successfully to identify changes in bone. Anesthetized animalswere examined and bone mineral content (BMC), BMC/LBM ratios, volumetricbone mineral density (vBMD), total body BMD, femur BMD and vertebra BMDwere measured.

The mouse was anesthetized by intraperitoneal injection of Avertin(1.25% 2,2,2,-tribromoethanol, 20 ml/kg body weight), body length andweight were measured, and then the mouse was placed in a prone positionon the platform of the PIXImus™ Densitometer (Lunar Inc.) for a DEXAscan. Using Lunar PIXImus software, the bone mineral density (BMD) andfat composition (% fat) and total tissue mass (TTM) were determined inthe regions of interest (ROI) [i.e., whole body, vertebrae, and bothfemurs].

Results:

The male (−/−) mice exhibited increased total tissue mass and total bodyfat (% and g) when compared to their gender matched wild-type (+/+)littermates and historical mean.

These studies suggest that mutant (−/−) non-human transgenic animalsexhibit a negative phenotype that would be associated with obesity.Thus, PRO34778 polypeptides or agonists thereof are essential for normalgrowth and metabolic processes and especially would be important in theprevention and/or treatment of obesity.

(e) Phenotypic Analysis: CNS/Neurology

In the area of neurology, analysis focused herein on identifying in vivovalidated targets for the treatment of neurological and psychiatricdisorders including depression, generalized anxiety disorders, attentiondeficit hyperactivity disorder, obsessive compulsive disorder,schizophrenia, cognitive disorders, hyperalgesia and sensory disorders.Neurological disorders include the category defined as “anxietydisorders” which include but are not limited to: mild to moderateanxiety, anxiety disorder due to a general medical condition, anxietydisorder not otherwise specified, generalized anxiety disorder, panicattack, panic disorder with agoraphobia, panic disorder withoutagoraphobia, posttraumatic stress disorder, social phobia, specificphobia, substance-induced anxiety disorder, acute alcohol withdrawal,obsessive compulsive disorder, agoraphobia, bipolar disorder I or II,bipolar disorder not otherwise specified, cyclothymic disorder,depressive disorder, major depressive disorder, mood disorder,substance-induced mood disorder. In addition, anxiety disorders mayapply to personality disorders including but not limited to thefollowing types: paranoid, antisocial, avoidant behavior, borderlinepersonality disorders, dependent, histronic, narcissistic,obsessive-compulsive, schizoid, and schizotypal.

Procedure:

Behavioral screens were performed on a cohort of 4 wild type, 4heterozygous and 8 homozygous mutant mice. All behavioral tests weredone between 12 and 16 weeks of age unless reduced viabilitynecessitates earlier testing. These tests included open field to measureanxiety, activity levels and exploration.

Functional Observational Battery (FOB) Test—Stress-Induced Hyperthermia:

The FOB is a series of situations applied to the animal to determinegross sensory and motor deficits. A subset of tests from the Irwinneurological screen that evaluates gross neurological function is used.In general, short-duration, tactile, olfactory, and visual stimuli areapplied to the animal to determine their ability to detect and respondnormally. These simple tests take approximately 10 minutes and the mouseis returned to its home cage at the end of testing.

Results:

Anxiety: The (−/−) mice exhibited increased sensitivity tostress-induced hyperthermia when compared with their gender-matched(+/+) littermates and the historical mean, suggesting an increasedanxiety-like response in the mutants. In summary, the functionalobservation testing revealed a phenotype associated with increasedanxiety which could be associated with mild to moderate anxiety, anxietydue to a general medical condition, and/or bipolar disorders;hyperactivity; sensory disorders; obsessive-compulsive disorders,schizophrenia or a paranoid personality. Thus, PRO34778 polypeptides oragonists thereof would be useful in the treatment of such neurologicaldisorders.48.48. Generation and Analysis of Mice Comprising DNA165608 (UNQ6208)Gene Disruptions

In these knockout experiments, the gene encoding PRO20233 polypeptides(designated as DNA165608) (UNQ6208) was disrupted. The gene specificinformation for these studies is as follows: the mutated mouse genecorresponds to nucleotide reference: NM_(—)178257 ACCESSION:NM_(—)178257 NID: gi 30142708 ref NM_(—)178257.1 Mus musculusinterleukin 22 receptor, alpha 1 (Il22ra1); protein reference: Q80XZ4ACCESSION: Q80XZ4 NID: Mus musculus (Mouse). Interleukin-22 receptoralpha chain; the human gene sequence reference: NM_(—)021258 ACCESSION:NM_(—)021258 NID: gi 31317238 refNM_(—)021258.2 Homo sapiens interleukin22 receptor, alpha 1 (IL22RA1); the human protein sequence correspondsto reference: Q9HB22 ACCESSION: Q9HB22 NID: Homo sapiens (Human). IL-22receptor.

The mouse gene of interest is Il22ra1 (interleukin 22 receptor, alpha1), ortholog of human IL22RA1. Aliases include Il22r, IL-22R, CRF2-9,and interleukin-22 receptor alpha chain.

IL22RA1 is a type I integral plasma membrane protein that functions as asubunit of IL22 receptor complex. IL22 receptor complex consists of bothIL22RA1 and interleukin 10 receptor beta (IL10RB), which bind withinterleukin 22 (IL22) released by T-cells (Xie et al, J Biol Chem275(40):31335-9 (2000); Kotenko et al, J Biol Chem 276(4):2725-32(2001)). Activation of the IL22 receptor complex can stimulate genetranscription through the JAK/STAT pathways and can activate several MAPkinase pathways (Xie et al, J Biol Chem 275(40):31335-9 (2000); Aggarwalet al, J Interferon Cytokine Res 21(12): 1047-53 (2001); Lejeune et al,J Biol Chem 277(37): 33676-82 (2002)). IL22RA1 is expressed in liver,kidney, pancreas, small intestine, colon, vascular endothelium, and skinkeratinocytes (Kotenko et al, J Biol Chem 276(4):2725-32 (2001);Aggarwal et al, J Interferon Cytokine Res 21(12):1047-53 (2001); Rameshet al, Cancer Res 63(16):5105-13 (2003); Wolk et al, Immunity21(2):241-54 (2004). Moreover, IL22RA1 expression is upregulated inliver in response to stimulation with lipopolysaccharides (Tachiiri etal, Genes Immun 4(2):153-9 (2003)) and in keratinocytes in response tointerferon-gamma (Wolk et al, Immunity 21(2):241-54 (2004)). IL22RA1 mayplay a role in innate immunity (Wolk et al, Immunity 21(2):241-54(2004)), prevention and repair of liver injury (Radaeva et al,Hepatology 39(5):13 32-42 (2004)), angiogenesis (Ramesh et al, CancerRes 63(6):5105-13 (2003)), and apoptosis of cancer cells (Sauane et al,J Cell Physiol 196(2):334-45 (2003)).

Targeted or gene trap mutations are generated in strain129SvEv^(Brd)-derived embryonic stem (ES) cells. The chimeric mice arebred to C57BL/6J albino mice to generate F1 heterozygous animals. Theseprogeny are intercrossed to generate F2 wild type, heterozygous, andhomozygous mutant progeny. On rare occasions, for example when very fewF1 mice are obtained from the chimera, F1 heterozygous mice are crossedto 129SvEv^(Brd)/C57 hybrid mice to yield additional heterozygousanimals for the intercross to generate the F2 mice. Level I phenotypicanalysis is performed on mice from this generation

wt het hom Total Observed 17 37 13 67 Expected 16.75 33.5 16.75 67Chi-Sq. = 0.46 Significance = 0.7945336 (hom/n) = 0.23 Avg. Litter Size= 7Mutation InformationMutation Type: Homologous Recombination (standard)Description: Coding exons 2 through 4 were targeted (NCBI accessionNM_(—)178257.1).1. Wild-type Expression Panel: Expression of the target gene wasdetected in embryonic stem (ES) cells and in all 13 adult tissue samplestested by RT-PCR, except skeletal muscle and bone.2. QC Expression: QC Images: Disruption of the target gene was confirmedby Southern hybridization analysis.

48.48.1. Phenotypic Analysis (for Disrupted Gene: DNA165608 (UNQ6208)

(a) Overall Phenotypic Summary:

Mutation of the gene encoding the ortholog of human interleukin 22receptor, alpha 1 (IL22RA1) resulted in small female (−/−) mice. Thefemale homozygous mutant mice were smaller than their gender-matchedwild-type littermates, exhibiting decreased body weight and length,decreased total tissue mass, and decreased lean body mass as well asdecreased bone mineral content and bone mineral density measurements.Disruption of the target gene was confirmed by Southern hybridizationanalysis.

(b) Expression

UNQ6208 is overexpressed in pancreatic tumors. [See EXAMPLES 54 and 55for protocol]

(c) Bone Metabolism & Body Diagnostics

(1) Tissue Mass & Lean Body Mass Measurements—Dexa

Dexa Analysis—Test Description:

Procedure: A cohort of 4 wild type, 4 heterozygous and 8 homozygous micewere tested in this assay. Dual Energy X-ray Absorptiometry (DEXA) hasbeen used successfully to identify changes in total tissue mass (TTM).

The mouse was anesthetized by intraperitoneal injection of Avertin(1.25% 2,2,2,-tribromoethanol, 20 ml/kg body weight), body length andweight were measured, and then the mouse was placed in a prone positionon the platform of the PIXImus™ Densitometer (Lunar Inc.) for a DEXAscan. Using Lunar PIXImus software, the bone mineral density (BMD) andfat composition (% fat) and total tissue mass (TTM) were determined inthe regions of interest (ROI, i.e., whole body, vertebrae, and bothfemurs).

Body Measurements (Body Length & Weight):

Body Measurements: A measurement of body length and weight was performedat approximately 16 weeks of age.

Results:

Weight: The (−/−) mice exhibited decreased mean body weight whencompared with that of their (+/+) littermates and the historical mean,the difference being more notable in the females.

Length: The female (−/−) mice exhibited decreased mean body length whencompared with that of their (+/+) littermates and the historical mean.

(2) Bone Metabolism: Radiology Phenotypic Analysis

In the area of bone metabolism, targets were identified herein for thetreatment of arthritis, osteoporosis, osteopenia and osteopetrosis aswell as identifying targets that promote bone healing. Tests included:

DEXA for measurement of bone mineral density on femur and vertebra

MicroCT for very high resolution and very high sensitivity measurementsof bone mineral density for both trabecular and cortical bone.

Dexa Analysis—Test Description:

Procedure: A cohort of 4 wild type, 4 heterozygous and 8 homozygous micewere tested in this assay. Dual Energy X-ray Absorptiometry (DEXA) hasbeen used successfully to identify changes in bone. Anesthetized animalswere examined and bone mineral content (BMC), BMC/LBM ratios, volumetricbone mineral density (vBMD), total body BMD, femur BMD and vertebra BMDwere measured.

The mouse was anesthetized by intraperitoneal injection of Avertin(1.25% 2,2,2,-tribromoethanol, 20 ml/kg body weight), body length andweight were measured, and then the mouse was placed in a prone positionon the platform of the PIXImus™ Densitometer (Lunar Inc.) for a DEXAscan. Using Lunar PIXImus software, the bone mineral density (BMD) andfat composition (% fat) and total tissue mass (TTM) were determined inthe regions of interest (ROI) [i.e., whole body, vertebrae, and bothfemurs].

Results:

DEXA: The female (−/−) mice exhibited notably decreased mean totaltissue mass, lean body mass, bone mineral content, and bone mineraldensity (total body BMD, femur BMD, and vertebrae BMD) when comparedwith that of their gender-matched (+/+) littermates and the historicalmeans.

Mutant female (−/−) mice deficient in the gene encoding PRO20233polypeptides show a phenotype consistent with growth retardation, markedby decreased body weight and length and total tissue mass and lean bodymass. Thus, antagonists or inhibitors of PRO20233 polypeptides or itsencoding gene would mimic these metabolic and growth related effects. Onthe other hand, PRO20233 polypeptides or agonists thereof would beuseful in the prevention and/or treatment of such metabolic disorders asdiabetes or other tissue wasting diseases.

In addition, the (−/−) mice analyzed by DEXA exhibited decreased bonemeasurements and decreased body mass measurements when compared withtheir (+/+) littermates, suggestive of abnormal bone disorders. The(−/−) mice exhibited a negative bone phenotype with abnormal decreasedbone measurements reflective of bone metabolic disorders. In addition,the decreased mean total tissue mass and lean body mass is indicative ofa metabolic disorder related to growth retardation and tissue wastingdisorders. The negative bone phenotype indicates that PRO20233polypeptides or agonists thereof would be useful for maintaining bonehomeostasis in addition to normal growth development. In addition,PRO20233 polypeptides would be useful in bone healing or for thetreatment of arthritis or osteoporosis, whereas antagonists (orinhibitors) of PRO20233 polypeptides or its encoding gene would lead toabnormal or pathological bone disorders including inflammatory diseasesassociated with abnormal bone metabolism including arthritis,osteoporosis and osteopenia.

48.49. Generation and Analysis of Mice Comprising DNA178511-2986(UNQ6973) Gene Disruptions

In these knockout experiments, the gene encoding PRO21956 polypeptides(designated as DNA178511-2986) (UNQ6973) was disrupted. The genespecific information for these studies is as follows: the mutated mousegene corresponds to nucleotide reference: NM_(—)011719 Mus musculuswingless-type MMTV integration site 9B (Wnt9b); protein reference:035468 Wnt-9b protein precursor (Wnt-15) (Wnt-14b)gi|18181917|dbj|BAB83866.1| Wnt14b [Mus musculus]; the human genesequence reference: NM_(—)003396 ACCESSION: NM_(—)003396 NID: gi17017975 refNM_(—)003396.1 Homo sapiens wingless-type MMTV integrationsite family, member 15 (WNT15); the human protein sequence correspondsto reference: 014905 ACCESSION:014905 NID: Homo sapiens (Human). WNT-15PROTEIN PRECURSOR (WNT-14B).

The mouse gene of interest is Wnt9b (wingless-type MMTV integration site9B), ortholog of human WNT9B (wingless-type MMTV integration sitefamily, member 9B). Aliases include Wnt14b, Wnt15, wingless-type MMTVintegration site 15, and wingless-type MMTV integration site familymember 15.

WNT9B is a secreted protein that likely functions as a ligand formembers of the frizzled family of G protein-coupled receptors (Katoh,Int J Mol Med 9(6):579-84 (2002)). The protein is expressed in mosttissues during development and in kidney and brain during adulthood(Qian et al, Genomics 81(1):34-46 (2003); Kirikoshi et al, int J Oncol19(5):947-52 (2001); Kirikoshi and Katoh, int J Mol Med 9(2):135-9(2002)). WNT9B may play a role in embryogenesis and neuronaldifferentiation (Kirikoshi et al, Int J Oncol 19(5):947-52 (2001);Kirikoshi and Katoh, Int J Mol Med 9(2):135-9 (2002)). Overexpression ofWNT9B may play a role in certain types of mammary cancer (Qian et al,Genomics 81(1):34-46 (2003)), and disruption of the WNT9B gene may causecleft lip and palate in mice (Juriloff et al, Birth Defects Res A ClinMol Teratol 73(2):103-13 (2005)).

Targeted or gene trap mutations are generated in strain129SvEv^(Brd)-derived embryonic stem (ES) cells. The chimeric mice arebred to C57BL/6J albino mice to generate F1 heterozygous animals. Theseprogeny are intercrossed to generate F2 wild type, heterozygous, andhomozygous mutant progeny. On rare occasions, for example when very fewF1 mice are obtained from the chimera, F1 heterozygous mice are crossedto 129SvEv^(Brd)/C57 hybrid mice to yield additional heterozygousanimals for the intercross to generate the F2 mice. Level I phenotypicanalysis is performed on mice from this generation

wt het hom Total Observed 19 38 0 57 Expected 14.25 28.5 14.2 57 Chi-Sq.= 15.61 Significance = 4.076915E−4 (hom/n) = 0.09 Avg. Litter Size = 8Mutation InformationMutation Type: Homologous Recombination (standard)Description: Coding exons 2 through 4 were targeted (NCBI accessionNM_(—)011719.2).1. Wild-type Expression Panel: Expression of the target gene wasdetected in embryonic stem (ES) cells and in all 13 adult tissue samplestested by RT-PCR, except lung; skeletal muscle; bone; and stomach, smallintestine, and colon.2. QC Expression: QC Images: Disruption of the target gene was confirmedby Southern hybridization analysis.

48.49.1. Phenotypic Analysis (for Disrupted Gene: DNA178511-2986(UNQ6973)

(a) Overall Phenotypic Summary:

Mutation of the gene encoding the ortholog of human wingless-type MMTVintegration site family, member 9B (WNT9B) resulted in lethality of(−/−) mutants. Genetic data indicate that this mutation resulted inlethality of the homozygous mutants. No notable phenotype was observedfor the heterozygous mice. Disruption of the target gene was confirmedby Southern hybridization analysis.

(b) Pathology

Microscopic: At 12.5 days, 41 embryos were observed: 10 (−/−) embryos,18 (+/−) embryos, 7 (+/+) embryos, 5 resorption moles, and 1inconclusive. However, no structural developmental abnormalities weredetected in the (−/−) embryos.

Gene Expression: LacZ activity was not detected in the panel of tissuesby immunohistochemical analysis.

Discussion Related to Embryonic Developmental Abnormality of Lethality:

Embryonic lethality in knockout mice usually results from variousserious developmental problems including but not limited toneurodegenerative diseases, angiogenic disorders, inflammatory diseases,or where the gene/protein has an important role in basic cell signalingprocesses in many cell types. In addition, embryonic lethals are usefulas potential cancer models. Likewise, the corresponding heterozygous(+/−) mutant animals are particularly useful when they exhibit aphenotype and/or a pathology report which reveals highly informativeclues as to the function of the knocked-out gene. For instance, EPOknockout animals were embryonic lethals, but the pathology reports onthe embryos showed a profound lack of RBCs.

48.50. Generation and Analysis of Mice Comprising DNA269238 (UNQ8782)Gene Disruptions

In these knockout experiments, the gene encoding PRO57290 polypeptides(designated as DNA269238) (UNQ8782) was disrupted. The gene specificinformation for these studies is as follows: the mutated mouse genecorresponds to nucleotide reference: NM_(—)032398 ACCESSION:NM_(—)032398 NID: gi 14161697 ref NM_(—)032398.1 Mus musculusplasmalemma vesicle associated protein (Plvap); protein reference:Q99JB1 ACCESSION: Q99JB1 NID: Mus musculus (Mouse). PV1 protein; thehuman gene sequence reference: NM_(—)031310 ACCESSION: NM_(—)031310 NID:gi13775237 refNM_(—)031310.1 Homo sapiens plasmalemma vesicle associatedprotein (PLVAP); the human protein sequence corresponds to reference:Q9BX97 ACCESSION: Q9BX97 NID: Homo sapiens (Human). PV1 protein.

The mouse gene of interest is Plvap (plasmalemma vesicle associatedprotein), ortholog of human PLVAP. Aliases include PV-1, MECA32, PV1,FELS, gp68, and fenestrated-endothelial linked structure protein.

PLVAP is a type II integral plasma membrane protein that is associatedwith both the stomatal diaphragms of caveolae, transendothelialchannels, and vesiculovacuolar organelles as well as the diaphragms ofendothelial fenestrae. The protein likely plays a role in the formationand structure of these diaphragms, which function as a selective barrierfor solutes. PLVAP may play a role in processes such as blood brainbarrier development and microvascular permeability (Hallmann et al, DevDyn 202(4):325-32 (1995); Stan et al, Genomics 72(3):304-13 (2001);Stan, Am J Physiol Heart Circ Physiol 286(4):H1347-53 (2004)). PLVAP isalso expressed in a variety of endocrine and non-endocrine cells, suchas pancreatic islet delta cells, neural lobe pituicytes, corpus lutealcells, germ cells within the adult seminiferous tubule, interstitialcells of the neonatal testis, and the thecal cell layer of developingfollicles (Hnasko et al, J Endocrinol 175(3):649-61 (2002)).

Targeted or gene trap mutations are generated in strain129SvEv^(Brd)-derived embryonic stem (ES) cells. The chimeric mice arebred to C57BL/6J albino mice to generate F1 heterozygous animals. Theseprogeny are intercrossed to generate F2 wild type, heterozygous, andhomozygous mutant progeny. On rare occasions, for example when very fewF1 mice are obtained from the chimera, F1 heterozygous mice are crossedto 129SvEv^(Brd)/C57 hybrid mice to yield additional heterozygousanimals for the intercross to generate the F2 mice. Level I phenotypicanalysis is performed on mice from this generation

wt het hom Total Observed 22 42 18 82 Expected 20.5 41 20.5 82 Chi-Sq. =1.77 Significance = 0.41271418 (hom/n) = 0.22 Avg. Litter Size = 8Mutation InformationMutation Type Homologous Recombination (standard)Description: Coding exon 1 was targeted (NCBI accession NM_(—)032398.1).1. Wild-type Expression Panel: Expression of the target gene wasdetected in embryonic stem (ES) cells and in all 13 adult tissue samplestested by RT-PCR, except bone.2. QC Expression: QC Images: Disruption of the target gene was confirmedby Southern hybridization analysis.

48.50.1. Phenotypic Analysis (for Disrupted Gene: DNA269238 (UNQ8782)

(a) Overall Phenotypic Summary:

Mutation of the gene encoding the ortholog of human plasmalemma vesicleassociated protein (PLVAP) resulted in the mutant (−/−) mice exhibitingdecreased anxiety in open field testing. The mutant (−/−) mice alsoshowed decreased mean serum glucose levels. Gene disruption wasconfirmed by Southern blot.

(b) Expression

UNQ8782 is overexpressed in kidney clear cell carcinoma. [See EXAMPLES54 and 55 for protocol]

(c) Phenotypic Analysis: CNS/Neurology

In the area of neurology, analysis focused herein on identifying in vivovalidated targets for the treatment of neurological and psychiatricdisorders including depression, generalized anxiety disorders, attentiondeficit hyperactivity disorder, obsessive compulsive disorder,schizophrenia, cognitive disorders, hyperalgesia and sensory disorders.Neurological disorders include the category defined as “anxietydisorders” which include but are not limited to: mild to moderateanxiety, anxiety disorder due to a general medical condition, anxietydisorder not otherwise specified, generalized anxiety disorder, panicattack, panic disorder with agoraphobia, panic disorder withoutagoraphobia, posttraumatic stress disorder, social phobia, specificphobia, substance-induced anxiety disorder, acute alcohol withdrawal,obsessive compulsive disorder, agoraphobia, bipolar disorder I or II,bipolar disorder not otherwise specified, cyclothymic disorder,depressive disorder, major depressive disorder, mood disorder,substance-induced mood disorder. In addition, anxiety disorders mayapply to personality disorders including but not limited to thefollowing types: paranoid, antisocial, avoidant behavior, borderlinepersonality disorders, dependent, histronic, narcissistic,obsessive-compulsive, schizoid, and schizotypal.

Procedure:

Behavioral screens were performed on a cohort of 4 wild type, 4heterozygous and 8 homozygous mutant mice. All behavioral tests weredone between 12 and 16 weeks of age unless reduced viabilitynecessitates earlier testing. These tests included open field to measureanxiety, activity levels and exploration.

Open Field Test:

Several targets of known drugs have exhibited phenotypes in the openfield test. These include knockouts of the seratonin transporter, thedopamine transporter (Giros et al., Nature. 1996 Feb. 15;379(6566):606-12), and the GABA receptor (Homanics et al., Proc NatlAcad Sci USA. 1997 Apr. 15; 94(8):4143-8). An automated open-field assaywas customized to address changes related to affective state andexploratory patterns related to learning. First, the field (40×40 cm)was selected to be relatively large for a mouse, thus designed to pickup changes in locomotor activity associated with exploration. Inaddition, there were 4 holes in the floor to allow for nose-poking, anactivity specifically related to exploration. Several factors were alsodesigned to heighten the affective state associated with this test. Theopen-field test is the first experimental procedure in which the miceare tested, and the measurements that were taken were the subjects'first experience with the chamber. In addition, the open-field wasbrightly lit. All these factors will heighten the natural anxietyassociated with novel and open spaces. The pattern and extent ofexploratory activity, and especially the center-to-total distancetraveled ratio, may then be able to discern changes related tosusceptibility to anxiety or depression. A large arena (40 cm×40 cm,VersaMax animal activity monitoring system from AccuScan Instruments)with infrared beams at three different levels was used to recordrearing, hole poke, and locomotor activity. The animal was placed in thecenter and its activity was measured for 20 minutes. Data from this testwas analyzed in five, 4-minute intervals. The total distance traveled(cm), vertical movement number (rearing), number of hole pokes, and thecenter to total distance ratio were recorded.

The propensity for mice to exhibit normal habituation responses to anovel environment is assessed by determining the overall change in theirhorizontal locomotor activity across the 5 time intervals. Thiscalculated slope of the change in activity over time is determined usingnormalized, rather than absolute, total distance traveled. The slope isdetermined from the regression line through the normalized activity ateach of the 5 time intervals. Normal habituation is represented by anegative slope value.

Results:

The female (−/−) mice exhibited an increased median sum time-in-centerduring open field testing when compared with their gender-matched (+/+)littermates and the historical mean, suggesting a decreased anxiety-likeresponse in the mutants.

A notable difference was observed during open field activity testing.The female (−/−) mice exhibited an increased median sum time in thecenter area when compared with their gender-matched (+/+) littermates,which is indicative of a decreased anxiety-like response in the mutants.Thus, knockout mice demonstrated a phenotype consistent with depression,generalized anxiety disorders, cognitive disorders, hyperalgesia andsensory disorders and/or bipolar disorders. Thus, PRO57290 polypeptidesand agonists thereof would be useful for the treatment or ameliorationof the symptoms associated with depressive disorders.

(d) Phenotypic Analysis: Metabolism—Blood Chemistry

In the area of metabolism, targets may be identified for the treatmentof diabetes. Blood chemistry phenotypic analysis includes blood glucosemeasurements. The COBAS Integra 400 (mfr: Roche) was used for runningblood chemistry tests on the mice. In the area of metabolism, targetsmay be identified for the treatment of diabetes.

Results:

The male (−/−) mice also exhibited a decreased mean serum glucose levelwhich could be related to abnormal glucose metabolism and/or diabetes.

In these studies the mutant (−/−) mice showed a decreased serum glucoselevels which could be due to an increased insulin sensitivity. Thus,antagonists (inhibitors) to PRO57290 polypeptides or its encoding genewould be useful in the treatment of impaired glucose homeostasis.

48.51. Generation and Analysis of Mice Comprising DNA228002 (UNQ9128)Gene Disruptions

In these knockout experiments, the gene encoding PRO38465 polypeptides(designated as DNA228002) (UNQ9128) was disrupted. The gene specificinformation for these studies is as follows: the mutated mouse genecorresponds to nucleotide reference: NM_(—)027209 Mus musculusmembrane-spanning 4-domains, subfamily A, member 6B (Ms4a6b); proteinreference: Q99N09 Membrane-spanning 4-domains subfamily A member 6Bgi|13649409|gb|AAK37418.1|MS4A6B protein [Mus musculus]; the human genesequence reference: NM_(—)152852 Homo sapiens membrane-spanning4-domains, subfamily A, member 6A (MS4A6A), transcript variant 1; thehuman protein sequence corresponds to reference: Q9H2W1 ACCESSION:Q9H2W1 NID: Homo sapiens (Human). CDA01 (MS4A6A-POLYMORPH) (MS4A6Aprotein).

The mouse gene of interest is Ms4a6b (membrane-spanning 4-domains,subfamily A, member 6B), ortholog of human MS4A6A (membrane-spanning4-domains, subfamily A, member 6A). Aliases include 1810027D10Rik,CDA01, MS4A6, 4SPAN3, CD20L3, MST090, MSTP090, 4SPAN3.2, MGC22650,HAIRB-iso, MS4A6A-polymorph, CD20-like precursor, four-spantransmembrane protein 3.1, and four-span transmembrane protein 3.2.

MS4A6A is an integral plasma membrane protein that likely functions as acomponent of a signal-transducing receptor complex. The protein consistsof four transmembrane segments within a CD20/IgE Fc receptor betasubunit family domain. Proteins with this domain include cell surfacereceptor subunits CD20, high-affinity IgE receptor beta chain, and HTm4,which are expressed on hematopoietic cells. Variable expression ofMS4A6A was evident in some B-cell, myelomonocytic, and erythroleukemiacell lines (Liang and Tedder, Genomics 72(2):119-27 (2001); Ishibashi etal, Gene 264(1):87-93 (2001)).

Targeted or gene trap mutations are generated in strain129SvEv^(Brd)-derived embryonic stem (ES) cells. The chimeric mice arebred to C57BL/6J albino mice to generate F1 heterozygous animals. Theseprogeny are intercrossed to generate F2 wild type, heterozygous, andhomozygous mutant progeny. On rare occasions, for example when very fewF1 mice are obtained from the chimera, F1 heterozygous mice are crossedto 129SvEv^(Brd)/C57 hybrid mice to yield additional heterozygousanimals for the intercross to generate the F2 mice. Level I phenotypicanalysis is performed on mice from this generation

wt het hom Total Observed 25 35 18 78 Expected 19.5 39 19.5 78 Chi-Sq. =0.21 Significance = 0.9003245 (hom/n) = 0.25 Avg. Litter Size = 9Mutation InformationMutation Type: Homologous Recombination (standard)Description: Coding exons 1 through 3 were targeted (NCBI accessionNM_(—)027209.2).1. Wild-type Expression Panel: Expression of the target gene wasdetected in all 26 adult tissue samples tested by RT-PCR, except boneand asthmatic lung.2. QC Expression: Disruption of the target gene was confirmed bySouthern hybridization analysis.

48.51.1. Phenotypic Analysis (for Disrupted Gene: DNA228002 (UNQ9128)

(a) Overall Phenotypic Summary:

Mutation of the gene encoding the ortholog of human membrane-spanning4-domains, subfamily A, member 6A (MS4A6A) resulted in the (−/−) miceexhibiting decreased insulin levels accompanied by increase mean serumglucose and an impaired glucose tolerance. The mutant (−/−) mice alsoshowed decreased skin fibroblast proliferation. Gene disruption wasconfirmed by Southern blot.

(b) Expression

UNQ9128 is overexpressed in ovarian tumors (serous cystadenocarcinomaincluding papillary). [See EXAMPLES 54 and 55 for protocol]

(c) Blood Chemistry/Glucose Tolerance

In the area of metabolism, targets may be identified for the treatmentof diabetes. Blood chemistry phenotypic analysis includes blood glucosemeasurements. The COBAS Integra 400 (mfr: Roche) was used for runningblood chemistry tests on the mice. In the area of metabolism, targetsmay be identified for the treatment of diabetes. Blood chemistryphenotypic analysis includes glucose tolerance tests to measure insulinsensitivity and changes in glucose metabolism. Abnormal glucosetolerance test results may indicate but may not be limited to thefollowing disorders or conditions: Diabetes Type 1 and Type 2, SyndromeX, various cardiovascular diseases and/or obesity.

Procedure: A cohort of 2 wild type and 4 homozygous mice were used inthis assay. The glucose tolerance test is the standard for definingimpaired glucose homeostasis in mammals. Glucose tolerance tests wereperformed using a Lifescan glucometer. Animals were injected IP at 2g/kg with D-glucose delivered as a 20% solution and blood glucose levelswere measured at 0, 30, 60 and 90 minutes after injection.

Results:

Blood Glucose Levels/Glucose Tolerance Test:

The (−/−) mice exhibited impaired glucose tolerance when compared withtheir gender-matched (+/+) littermates and the historical means. The(−/−) mice also exhibited an increased mean fasting serum glucose level.

These studies indicated that (−/−) mice exhibit a decreased or impairedglucose tolerance in the presence of normal fasting glucose at all 3intervals tested when compared with their gender-matched (+/+)littermates and the historical means. Thus, knockout mutant miceexhibited the phenotypic pattern of an impaired glucose homeostasis, andtherefor PRO38496 polypeptides (or agonists thereof) or its encodinggene would be useful in the treatment of conditions associated with animpaired glucose homeostasis and/or various cardiovascular diseases,including diabetes.

Insulin Data:

Test Description: Lexicon Genetics uses the Cobra II Series Auto-GammaCounting System in its clinical settings for running quantitativeInsulin assays on mice.

Results:

The (−/−) mice exhibited a decreased mean serum insulin level whencompared with their gender-matched (+/+) littermates and the historicalmean.

Serum Glucose Levels

Results:

The homozygous (−/−) mice exhibited increased mean serum glucose levelswhen compared with that of their gender-matched (+/+) littermates andthe historical mean.

Thus, the mutant (−/−) mice exhibited hyperglycemia which is associatedwith an altered glucose metabolism or diabetes. PRO38465 polypeptides oragonists thereof would be useful in maintaining normal glucoselevels/metabolism and possibly useful in the treatment of diabetes.These results are consistent with the observed decrease in insulinlevels in the mutant (−/−) mice.

(d) Adult Skin Cell Proliferation:

Procedure: Skin cells were isolated from 16 week old animals (2 wildtype and 4 homozygous mice). These were developed into primaryfibroblast cultures and the fibroblast proliferation rates were measuredin a strictly controlled protocol. The ability of this assay to detecthyper-proliferative and hypo-proliferative phenotypes has beendemonstrated with p53 and Ku80. Proliferation was measured using Brduincorporation.

Specifically, in these studies the skin fibroblast proliferation assaywas used. An increase in the number of cells in a standardized culturewas used as a measure of relative proliferative capacity. Primaryfibroblasts were established from skin biopsies taken from wild type andmutant mice. Duplicate or triplicate cultures of 0.05 million cells wereplated and allowed to grow for six days. At the end of the cultureperiod, the number of cells present in the culture was determined usinga electronic particle counter.

Results:

The female (−/−) mice exhibited a decreased mean skin fibroblastproliferation rate when compared with their gender-matched (+/+)littermates.

Thus, homozygous mutant mice demonstrated a hypo-proliferativephenotype. As suggested by these observations, antagonists or inhibitorsof PRO38465 polypeptides would mimic this hypo-proliferative phenotypeand could function as tumor suppressors and would be useful indecreasing abnormal cell proliferation.

48.52. Generation and Analysis of Mice Comprising DNA228199 (UNQ9638)Gene Disruptions

In these knockout experiments, the gene encoding PRO38683 polypeptides(designated as DNA228199) (UNQ9638) was disrupted. The gene specificinformation for these studies is as follows: the mutated mouse genecorresponds to nucleotide reference: XM_(—)357986 PREDICTED: Musmusculus RIKEN cDNA 1110008114 gene (1110008114Rik); protein reference:XP_(—)357986 MUC16 [Mus musculus]; the human gene sequence reference:AF414-442 Homo sapiens ovarian cancer related tumor marker CA125 mRNA,complete cds; the human protein sequence corresponds to reference:Q8WX17 ACCESSION: Q8WX17 NID: Homo sapiens (Human). Ovarian cancerrelated tumor marker CA125.

The mouse gene of interest is RIKEN cDNA 1110008114 gene, ortholog ofhuman MUC16 (mucin 16). Aliases include CA125, FLJ14303, and CA125ovarian cancer antigen.

MUC16 is a giant integral plasma membrane glycoprotein expressed in avariety of epithelia and over-expressed in epithelial ovarian cancercells. The protein consists of a large N-terminal extracellular segment,a transmembrane segment, and a short C-terminal cytoplasmic domain. Theextracellular N-terminal segment varies in length due to alternativesplicing but can consist of as many as 20,000 amino acids. This segmentis heavily O-glycosylated, containing many serine and threonineresidues. The extracellular segment consists of an N-terminal domain andas many as 40-60 tandem repeats of SEA domains (domain found in seaurchin sperm protein, enterokinase, agrin) near the plasma membrane. SEAdomains are generally found in heavily glycosylated proteins and arelikely involved in binding with carbohydrate side chains on neighboringmolecules (SMART accession SM00200). The extracellular segment of MUC16can be released into the extracellular space by proteolytic cleavage(O'Brien et al, Tumour Biol 22(6):348-66 (2001); O'Brien et al, TumourBiol 23(3):154-69 (2002)). MUC16 is likely involved in immunesuppression and reproduction, protecting the embryo from the maternalimmune response. Upregulation of MUC16 in epithelial ovarian tumor cellsmay enable escape from cytotoxic T-cells and natural killer cells (KuiWong et al, J Biol Chem 278(31):28619-34 (2003)). Moreover, MUC16 mayplay a role in heterotypic cell adhesion and metastasis (Rump et al, JBiol Chem 279(10):9190-8 (2004)).

Targeted or gene trap mutations are generated in strain129SvEv^(Brd)-derived embryonic stem (ES) cells. The chimeric mice arebred to C57BL/6J albino mice to generate F1 heterozygous animals. Theseprogeny are intercrossed to generate F2 wild type, heterozygous, andhomozygous mutant progeny. On rare occasions, for example when very fewF1 mice are obtained from the chimera, F1 heterozygous mice are crossedto 129SvEv^(Brd)/C57 hybrid mice to yield additional heterozygousanimals for the intercross to generate the F2 mice. Level I phenotypicanalysis is performed on mice from this generation

wt het hom Total Observed 16 41 21 78 Expected 19.5 39 19.5 78 Chi-Sq. =1.26 Significance = 0.5325918 (hom/n) = 0.22 Avg. Litter Size = 9Mutation InformationMutation Type Homologous Recombination (standard)Description: Coding exons 60-63 were targeted (NCBI accessionXM_(—)357986.2).1. Wild-type Expression Panel: Expression of the target gene wasdetected in embryonic stem (ES) cells and in all 13 adult tissue samplestested by RT-PCR, except brain, skeletal muscle, and bone. FurtherRT-PCR studies showed expression in normal RNA tissue derived from (+/+)mice as follows: male (+/+) mice in heart, lung, testis and vasdeferens; female (+/+) mice heart, lung, ovary/oviduct (includingfallopian tube), and uterus.2. QC Expression: Disruption of the target gene was confirmed bySouthern hybridization analysis.

48.52.1. Phenotypic Analysis (for Disrupted Gene: DNA228199 (UNQ9638)

(a) Overall Phenotypic Summary:

Mutation of the gene encoding the ortholog of human mucin 16 (MUC16)resulted in the (−/−) mice exhibiting a decreased skin fibroblastproliferation rate. Gene disruption was confirmed by Southern blot.

(b) Adult Skin Cell Proliferation:

Procedure: Skin cells were isolated from 16 week old animals (2 wildtype and 4 homozygous mice). These were developed into primaryfibroblast cultures and the fibroblast proliferation rates were measuredin a strictly controlled protocol. The ability of this assay to detecthyper-proliferative and hypo-proliferative phenotypes has beendemonstrated with p53 and Ku80. Proliferation was measured using Brduincorporation.

Specifically, in these studies the skin fibroblast proliferation assaywas used. An increase in the number of cells in a standardized culturewas used as a measure of relative proliferative capacity. Primaryfibroblasts were established from skin biopsies taken from wild type andmutant mice. Duplicate or triplicate cultures of 0.05 million cells wereplated and allowed to grow for six days. At the end of the cultureperiod, the number of cells present in the culture was determined usinga electronic particle counter.

Results: The female (−/−) mice exhibited a decreased mean skinfibroblast proliferation rate when compared with their gender-matched(+/+) littermates.

Thus, homozygous mutant mice demonstrated a hypo-proliferativephenotype. As suggested by these observations, antagonists or inhibitorsof PRO38683 polypeptides would mimic this hypo-proliferative phenotypeand could function as tumor suppressors and would be useful indecreasing abnormal cell proliferation.

48.53. Generation and Analysis of Mice Comprising DNA329632 (UNQ16168)Gene Disruptions

In these knockout experiments, the gene encoding PRO85161 polypeptides(designated as DNA329632) (UNQ16168) was disrupted. The gene specificinformation for these studies is as follows: the mutated mouse genecorresponds to nucleotide reference: NM_(—)027022 Mus musculuschemokine-like factor super family 2A (Cklfsf2a); protein reference:Q9DAR1 ACCESSION: Q9DAR1 NID: Mus musculus (Mouse). 1700001K04Rikprotein; the human gene sequence reference: NM_(—)144673 Homo sapienschemokine-like factor super family 2 (CKLFSF2); the human proteinsequence corresponds to reference: Q8TAZ6 ACCESSION: Q8TAZ6 NID: Homosapiens (Human). Similar to putative (Chemokine-like factor super family2).

The mouse gene of interest is Cklfsf2a (chemokine-like factor superfamily 2A), ortholog of human CKLFSF2 (chemokine-like factor superfamily 2). Aliases include C32, Cklf, ARR19, CKLF3, CKLF4, CKLF5, Cklf1,UCK-1, Cklfsf2-1b, 1700001K04Rik, 1700041N15Rik, 1700063K20Rik,chemokine-like factor, chemokine-like factor superfamily 2-1b, FLJ25732,MGC39436, and CKLFSF2-v2.

CKLFSF2 is an integral membrane protein expressed primarily in testisand prostate. The protein contains four transmembrane segments within aMARVEL (membrane-associating) domain. Proteins with MARVEL domains mayfunction in membrane apposition events, such as transport vesiclebiogenesis (PFAM accession PF01284). CKLFSF2 is located in the cytoplasmbut translocates to the nucleus after forming a complex withandrogen-activated androgen receptors. CKLFSF2 is capable of repressingandrogen receptor transactivation by recruiting histone deacetylase 4.Thus, CKLFSF2 appears to function as an androgen receptor corepressor.Bioinformatic analyses suggest that CKLFSF2 is located in the plasmamembrane. CKLFSF2 may be involved in cell proliferation, celldifferentiation, and male reproductive processes (Xia et al, BiochimBiophys Acta 1591 (1-3):163-173 (2002); Rui et al, Mol Biol Rep30(4):229-37 (2003); Han et al, Genomics 81(6):609-17 (2003); Jeong etal, Mol Endocrinol 18(1):13-25 (2004)).

Targeted or gene trap mutations are generated in strain129SvEv^(Brd)-derived embryonic stem (ES) cells. The chimeric mice arebred to C57BL/6J albino mice to generate F1 heterozygous animals. Theseprogeny are intercrossed to generate F2 wild type, heterozygous, andhomozygous mutant progeny. On rare occasions, for example when very fewF1 mice are obtained from the chimera, F1 heterozygous mice are crossedto 129SvEv^(Brd)/C57 hybrid mice to yield additional heterozygousanimals for the intercross to generate the F2 mice. Level I phenotypicanalysis is performed on mice from this generation

wt het hom Total Observed 12 27 13 52 Expected 13 26 13 52 Chi-Sq. =2.41 Significance = 0.29969198 (hom/n) = 0.25 Avg. Litter Size = 8Mutation InformationMutation Type: Homologous Recombination (standard)Description: Coding exons 1 and 2 were targeted (NCBI accessionNM_(—)027022.2).1. Wild-type Expression Panel: Expression of the target gene wasdetected only in brain among the 13 adult tissue samples tested byRT-PCR.2. QC Expression: QC Images: Disruption of the target gene was confirmedby Southern hybridization analysis.

48.53.1. Phenotypic Analysis (for Disrupted Gene: DNA329632 (UNQ16168)

(a) Overall Phenotypic Summary:

Mutation of the gene encoding the ortholog of human chemokine-likefactor super family 2 (CKLFSF2) resulted in the (−/−) mice exhibitingincreased alkaline phosphatase levels and increased body fat. Genedisruption was confirmed by Southern blot.

(b) Phenotypic Analysis: Metabolism—Blood Chemistry

In the area of metabolism, targets may be identified for the treatmentof diabetes. Blood chemistry phenotypic analysis includes blood glucosemeasurements. The COBAS Integra 400 (mfr: Roche) was used for runningblood chemistry tests on the mice. In addition to measuring bloodglucose levels the following blood chemistry tests are also routinelyperformed: Alkaline Phosphatase; Alanine Amino-Transferase; Albumin;Bilirubin; Phosphorous; Creatinine; BUN=Blood Urea Nitrogen; Calcium;Uric Acid; Sodium; Potassium; and Chloride. In the area of metabolism,targets may be identified for the treatment of diabetes. Blood chemistryphenotypic analysis includes glucose tolerance tests to measure insulinsensitivity and changes in glucose metabolism. Abnormal glucosetolerance test results may indicate but may not be limited to thefollowing disorders or conditions: Diabetes Type 1 and Type 2, SyndromeX, various cardiovascular diseases and/or obesity.

Results:

The male (−/−) mice exhibited increased mean serum alkaline phosphatasewhen compared with their gender-matched (+/+) littermates and thehistorical means.

(c) Bone Metabolism & Radiology Phenotypic Analysis

In the area of bone metabolism, targets were identified herein for thetreatment of arthritis, osteoporosis, osteopenia and osteopetrosis aswell as identifying targets that promote bone healing. Tests included:

DEXA for measurement of bone mineral density on femur and vertebra

MicroCT for very high resolution and very high sensitivity measurementsof bone mineral density for both trabecular and cortical bone.

Dexa Analysis—Test Description:

Procedure: A cohort of 4 wild type, 4 heterozygous and 8 homozygous micewere tested in this assay. Dual Energy X-ray Absorptiometry (DEXA) hasbeen used successfully to identify changes in bone. Anesthetized animalswere examined and bone mineral content (BMC), BMC/LBM ratios, volumetricbone mineral density (vBMD), total body BMD, femur BMD and vertebra BMDwere measured.

The mouse was anesthetized by intraperitoneal injection of Avertin(1.25% 2,2,2,-tribromoethanol, 20 ml/kg body weight), body length andweight were measured, and then the mouse was placed in a prone positionon the platform of the PIXImus™ Densitometer (Lunar Inc.) for a DEXAscan. Using Lunar PIXImus software, the bone mineral density (BMD) andfat composition (% fat) and total tissue mass (TTM) were determined inthe regions of interest (ROI) [i.e., whole body, vertebrae, and bothfemurs].

Results:

DEXA: The female (−/−) mice exhibited increased mean percent total bodyfat and total fat mass when compared with their gender-matched (+/+)littermates and the historical means.

These studies suggest that mutant (−/−) non-human transgenic animalsexhibit a negative phenotype that would be associated with obesity.Thus, PRO85161 polypeptides or agonists thereof are essential for normalgrowth and metabolic processes and especially would be important in theprevention and/or treatment of obesity.

Example 49

Use of PRO226, PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382,PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343,PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571,PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381,PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017,PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196, PRO34778,PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161 as ahybridization Probe

The following method describes use of a nucleotide sequence encoding aPRO226, PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382,PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343,PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571,PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381,PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017,PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196, PRO34778,PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161 polypeptideas a hybridization probe.

DNA comprising the coding sequence of full-length or mature PRO226,PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382, PRO444,PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343, PRO1379,PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571, PRO1572,PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381, PRO4407,PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017, PRO7174,PRO9744, PRO9821, PRO9852, PRO9873, PRO10196, PRO34778, PRO20233,PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161 polypeptides asdisclosed herein is employed as a probe to screen for homologous DNAs(such as those encoding naturally-occurring variants of PRO226, PRO257,PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382, PRO444, PRO705,PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343, PRO1379, PRO1380,PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571, PRO1572, PRO1759,PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381, PRO4407, PRO4425,PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017, PRO7174, PRO9744,PRO9821, PRO9852, PRO9873, PRO10196, PRO34778, PRO20233, PRO21956,PRO57290, PRO38465, PRO38683 or PRO85161 polypeptides) in human tissuecDNA libraries or human tissue genomic libraries.

Hybridization and washing of filters containing either library DNAs isperformed under the following high stringency conditions. Hybridizationof radiolabeled PRO226-, PRO257-, PRO268-, PRO290-, PRO36006-, PRO363-,PRO365-, PRO382-, PRO444-, PRO705-, PRO1071-, PRO1125-, PRO1134-,PRO1155-, PRO1281-, PRO1343-, PRO1379-, PRO1380-, PRO1387-, PRO1419-,PRO1433-, PRO1474-, PRO1550-, PRO1571-, PRO1572-, PRO1759-, PRO1904-,PRO35193-, PRO4341-, PRO4348-, PRO4369-, PRO4381-, PRO4407-, PRO4425-,PRO4985-, PRO4989-, PRO5737-, PRO5800-, PRO5993-, PRO6017-, PRO7174-,PRO9744-, PRO9821-, PRO9852-, PRO9873-, PRO10196-, PRO34778-, PRO20233-,PRO21956-, PRO57290-, PRO38465-, PRO38683- or PRO85161-derived probe tothe filters is performed in a solution of 50% formamide, 5×SSC, 0.1%SDS, 0.1% sodium pyrophosphate, 50 mM sodium phosphate, pH 6.8,2×Denhardt's solution, and 10% dextran sulfate at 42° C. for 20 hours.Washing of the filters is performed in an aqueous solution of 0.1×SSCand 0.1% SDS at 42° C.

DNAs having a desired sequence identity with the DNA encodingfull-length native sequence PRO226, PRO257, PRO268, PRO290, PRO36006,PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125, PRO1134,PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433,PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341,PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737,PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873,PRO10196, PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 orPRO85161 polypeptides can then be identified using standard techniquesknown in the art.

Example 50

Expression of PRO226, PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365,PRO382, PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281,PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550,PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369,PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993,PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196,PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161in E. coli

This example illustrates preparation of an unglycosylated form ofPRO226, PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382,PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343,PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571,PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381,PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017,PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196, PRO34778,PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161polypeptides by recombinant expression in E. coli.

The DNA sequence encoding a PRO226, PRO257, PRO268, PRO290, PRO36006,PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125, PRO1134,PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433,PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341,PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737,PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873,PRO10196, PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 orPRO85161 polypeptide is initially amplified using selected PCR primers.The primers should contain restriction enzyme sites which correspond tothe restriction enzyme sites on the selected expression vector. Avariety of expression vectors may be employed. An example of a suitablevector is pBR322 (derived from E. coli; see Bolivar et al., Gene, 2:95(1977)) which contains genes for ampicillin and tetracycline resistance.The vector is digested with restriction enzyme and dephosphorylated. ThePCR amplified sequences are then ligated into the vector. The vectorwill preferably include sequences which encode for an antibioticresistance gene, a trp promoter, a polyhis leader (including the firstsix STII codons, polyhis sequence, and enterokinase cleavage site), thePRO226, PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382,PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343,PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571,PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381,PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017,PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196, PRO34778,PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161 codingregion, lambda transcriptional terminator, and an argU gene.

The ligation mixture is then used to transform a selected E. coli strainusing the methods described in Sambrook et al., supra. Transformants areidentified by their ability to grow on LB plates and antibioticresistant colonies are then selected. Plasmid DNA can be isolated andconfirmed by restriction analysis and DNA sequencing.

Selected clones can be grown overnight in liquid culture medium such asLB broth supplemented with antibiotics. The overnight culture maysubsequently be used to inoculate a larger scale culture. The cells arethen grown to a desired optical density, during which the expressionpromoter is turned on.

After culturing the cells for several more hours, the cells can beharvested by centrifugation. The cell pellet obtained by thecentrifugation can be solubilized using various agents known in the art,and the solubilized PRO226, PRO257, PRO268, PRO290, PRO36006, PRO363,PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155,PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474,PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348,PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800,PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196,PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161protein can then be purified using a metal chelating column underconditions that allow tight binding of the protein.

PRO226, PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382,PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343,PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571,PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381,PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017,PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196, PRO34778,PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161 may beexpressed in E. coli in a poly-His tagged form, using the followingprocedure. The DNA encoding PRO226, PRO257, PRO268, PRO290, PRO36006,PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125, PRO1134,PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433,PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341,PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737,PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873,PRO10196, PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 orPRO85161 is initially amplified using selected PCR primers. The primerswill contain restriction enzyme sites which correspond to therestriction enzyme sites on the selected expression vector, and otheruseful sequences providing for efficient and reliable translationinitiation, rapid purification on a metal chelation column, andproteolytic removal with enterokinase. The PCR-amplified, poly-Histagged sequences are then ligated into an expression vector, which isused to transform an E. coli host based on strain 52 (W3110 fuhA (tonA)lon galE rpoHts (htpRts) clpP (lacIq). Transformants are first grown inLB containing 50 mg/ml carbenicillin at 30° C. with shaking until anO.D.600 of 3-5 is reached. Cultures are then diluted 50-100 fold intoCRAP media (prepared by mixing 3.57 g (NH₄)₂SO₄, 0.71 g sodiumcitrate.2H2O, 1.07 g KCl, 5.36 g Difco yeast extract, 5.36 g Sheffieldhycase SF in 500 mL water, as well as 110 mM MPOS, pH 7.3, 0.55% (w/v)glucose and 7 mM MgSO₄) and grown for approximately 20-30 hours at 30°C. with shaking. Samples are removed to verify expression by SDS-PAGEanalysis, and the bulk culture is centrifuged to pellet the cells. Cellpellets are frozen until purification and refolding.

E. coli paste from 0.5 to 1 L fermentations (6-10 g pellets) isresuspended in 10 volumes (w/v) in 7 M guanidine, 20 mM Tris, pH 8buffer. Solid sodium sulfite and sodium tetrathionate is added to makefinal concentrations of 0.1 M and 0.02 M, respectively, and the solutionis stirred overnight at 4° C. This step results in a denatured proteinwith all cysteine residues blocked by sulfitolization. The solution iscentrifuged at 40,000 rpm in a Beckman Ultracentifuge for 30 min. Thesupernatant is diluted with 3-5 volumes of metal chelate column buffer(6 M guanidine, 20 mM Tris, pH 7.4) and filtered through 0.22 micronfilters to clarify. The clarified extract is loaded onto a 5 ml QiagenNi-NTA metal chelate column equilibrated in the metal chelate columnbuffer. The column is washed with additional buffer containing 50 mMimidazole (Calbiochem, Utrol grade), pH 7.4. The protein is eluted withbuffer containing 250 mM imidazole. Fractions containing the desiredprotein are pooled and stored at 4° C. Protein concentration isestimated by its absorbance at 280 nm using the calculated extinctioncoefficient based on its amino acid sequence.

The proteins are refolded by diluting the sample slowly into freshlyprepared refolding buffer consisting of: 20 mM Tris, pH 8.6, 0.3 M NaCl,2.5 M urea, 5 mM cysteine, 20 mM glycine and 1 mM EDTA. Refoldingvolumes are chosen so that the final protein concentration is between 50to 100 micrograms/ml. The refolding solution is stirred gently at 4° C.for 12-36 hours. The refolding reaction is quenched by the addition ofTFA to a final concentration of 0.4% (pH of approximately 3). Beforefurther purification of the protein, the solution is filtered through a0.22 micron filter and acetonitrile is added to 2-10% finalconcentration. The refolded protein is chromatographed on a Poros R1/Hreversed phase column using a mobile buffer of 0.1% TFA with elutionwith a gradient of acetonitrile from 10 to 80%. Aliquots of fractionswith A280 absorbance are analyzed on SDS polyacrylamide gels andfractions containing homogeneous refolded protein are pooled. Generally,the properly refolded species of most proteins are eluted at the lowestconcentrations of acetonitrile since those species are the most compactwith their hydrophobic interiors shielded from interaction with thereversed phase resin. Aggregated species are usually eluted at higheracetonitrile concentrations. In addition to resolving misfolded forms ofproteins from the desired form, the reversed phase step also removesendotoxin from the samples.

Fractions containing the desired folded PRO226, PRO257, PRO268, PRO290,PRO36006, PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125,PRO1134, PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419,PRO1433, PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193,PRO4341, PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989,PRO5737, PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852,PRO9873, PRO10196, PRO34778, PRO20233, PRO21956, PRO57290, PRO38465,PRO38683 or PRO85161 polypeptide are pooled and the acetonitrile removedusing a gentle stream of nitrogen directed at the solution. Proteins areformulated into 20 mM Hepes, pH 6.8 with 0.14 M sodium chloride and 4%mannitol by dialysis or by gel filtration using G25 Superfine(Pharmacia) resins equilibrated in the formulation buffer and sterilefiltered.

Example 51

Expression of PRO226, PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365,PRO382, PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281,PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550,PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369,PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993,PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196,PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161in Mammalian Cells

This example illustrates preparation of a potentially glycosylated formof a PRO226, PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382,PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343,PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571,PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381,PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017,PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196, PRO34778,PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161 polypeptideby recombinant expression in mammalian cells.

The vector, pRK5 (see EP 307,247, published Mar. 15, 1989), is employedas the expression vector. Optionally, the PRO226, PRO257, PRO268,PRO290, PRO36006, PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071,PRO1125, PRO1134, PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387,PRO1419, PRO1433, PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904,PRO35193, PRO4341, PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985,PRO4989, PRO5737, PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821,PRO9852, PRO9873, PRO10196, PRO34778, PRO20233, PRO21956, PRO57290,PRO38465, PRO38683 or PRO85161 DNA is ligated into pRK5 with selectedrestriction enzymes to allow insertion of the PRO226, PRO257, PRO268,PRO290, PRO36006, PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071,PRO1125, PRO1134, PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387,PRO1419, PRO1433, PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904,PRO35193, PRO4341, PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985,PRO4989, PRO5737, PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821,PRO9852, PRO9873, PRO10196, PRO34778, PRO20233, PRO21956, PRO57290,PRO38465, PRO38683 or PRO85161 DNA using ligation methods such asdescribed in Sambrook et al., supra. The resulting vector is calledpRK5-PRO226, pRK5-PRO257, pRK5-PRO268, pRK5-PRO290, pRK5-PRO36006,pRK5-PRO363, pRK5-PRO365, pRK5-PRO382, pRK5-PRO444, pRK5-PRO705,pRK5-PRO1071, pRK5-PRO1125, pRK5-PRO1134, pRK5-PRO1155, pRK5-PRO1281,pRK5-PRO1343, pRK5-PRO1379, pRK5-PRO1380, pRK5-PRO1387, pRK5-PRO1419,pRK5-PRO1433, pRK5-PRO1474, pRK5-PRO1550, pRK5-PRO1571, pRK5-PRO1572,pRK5-PRO1759, pRK5-PRO1904, pRK5-PRO35193, pRK5-PRO4341, pRK5-PRO4348,pRK5-PRO4369, pRK5-PRO4381, pRK5-PRO4407, pRK5-PRO4425, pRK5-PRO4985,pRK5-PRO4989, pRK5-PRO5737, pRK5-PRO5800, pRK5-PRO5993, pRK5-PRO6017,pRK5-PRO7174, pRK5-PRO9744, pRK5-PRO9821, pRK5-PRO9852, pRK5-PRO9873,pRK5-PRO10196, pRK5-PRO34778, pRK5-PRO20233, pRK5-PRO21956,pRK5-PRO57290, pRK5-PRO38465, pRK5-PRO38683 or pRK5-PRO85161.

The selected host cells may be 293 cells. Human 293 cells (ATCC CCL1573) are grown to confluence in tissue culture plates in medium such asDMEM supplemented with fetal calf serum and optionally, nutrientcomponents and/or antibiotics. About 10 μg pRK5-PRO226, pRK5-PRO257,pRK5-PRO268, pRK5-PRO290, pRK5-PRO36006, pRK5-PRO363, pRK5-PRO365,pRK5-PRO382, pRK5-PRO444, pRK5-PRO705, pRK5-PRO1071, pRK5-PRO1125,pRK5-PRO1134, pRK5-PRO1155, pRK5-PRO1281, pRK5-PRO1343, pRK5-PRO1379,pRK5-PRO1380, pRK5-PRO1387, pRK5-PRO1419, pRK5-PRO1433, pRK5-PRO1474,pRK5-PRO1550, pRK5-PRO1571, pRK5-PRO1572, pRK5-PRO1759, pRK5-PRO1904,pRK5-PRO35193, pRK5-PRO4341, pRK5-PRO4348, pRK5-PRO4369, pRK5-PRO4381,pRK5-PRO4407, pRK5-PRO4425, pRK5-PRO4985, pRK5-PRO4989, pRK5-PRO5737,pRK5-PRO5800, pRK5-PRO5993, pRK5-PRO6017, pRK5-PRO7174, pRK5-PRO9744,pRK5-PRO9821, pRK5-PRO9852, pRK5-PRO9873, pRK5-PRO10196, pRK5-PRO34778,pRK5-PRO20233, pRK5-PRO21956, pRK5-PRO57290, pRK5-PRO38465,pRK5-PRO38683 or pRK5-PRO85161 DNA is mixed with about 1 μg DNA encodingthe VA RNA gene [Thimmappaya et al., Cell, 31:543 (1982)] and dissolvedin 500 μl of 1 mM Tris-HCl, 0.1 mM EDTA, 0.227 M CaCl₂. To this mixtureis added, dropwise, 500 μl of 50 mM HEPES (pH 7.35), 280 mM NaCl, 1.5 mMNaPO₄, and a precipitate is allowed to form for 10 minutes at 25° C. Theprecipitate is suspended and added to the 293 cells and allowed tosettle for about four hours at 37° C. The culture medium is aspiratedoff and 2 ml of 20% glycerol in PBS is added for 30 seconds. The 293cells are then washed with serum free medium, fresh medium is added andthe cells are incubated for about 5 days.

Approximately 24 hours after the transfections, the culture medium isremoved and replaced with culture medium (alone) or culture mediumcontaining 200 μCi/ml ³⁵S-cysteine and 200 μCi/ml ³⁵S-methionine. Aftera 12 hour incubation, the conditioned medium is collected, concentratedon a spin filter, and loaded onto a 15% SDS gel. The processed gel maybe dried and exposed to film for a selected period of time to reveal thepresence of PRO226, PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365,PRO382, PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281,PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550,PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369,PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993,PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196,PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161polypeptides. The cultures containing transfected cells may undergofurther incubation (in serum free medium) and the medium is tested inselected bioassays.

In an alternative technique, PRO226, PRO257, PRO268, PRO290, PRO36006,PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125, PRO1134,PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433,PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341,PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737,PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873,PRO10196, PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 orPRO85161 may be introduced into 293 cells transiently using the dextransulfate method described by Somparyrac et al., Proc. Natl. Acad. Sci.,12:7575 (1981). 293 cells are grown to maximal density in a spinnerflask and 700 μg pRK5-PRO226, pRK5-PRO257, pRK5-PRO268, pRK5-PRO290,pRK5-PRO36006, pRK5-PRO363, pRK5-PRO365, pRK5-PRO382, pRK5-PRO444,pRK5-PRO705, pRK5-PRO1071, pRK5-PRO1125, pRK5-PRO1134, pRK5-PRO1155,pRK5-PRO1281, pRK5-PRO1343, pRK5-PRO1379, pRK5-PRO1380, pRK5-PRO1387,pRK5-PRO1419, pRK5-PRO1433, pRK5-PRO1474, pRK5-PRO1550, pRK5-PRO1571,pRK5-PRO1572, pRK5-PRO1759, pRK5-PRO1904, pRK5-PRO35193, pRK5-PRO4341,pRK5-PRO4348, pRK5-PRO4369, pRK5-PRO4381, pRK5-PRO4407, pRK5-PRO4425,pRK5-PRO4985, pRK5-PRO4989, pRK5-PRO5737, pRK5-PRO5800, pRK5-PRO5993,pRK5-PRO6017, pRK5-PRO7174, pRK5-PRO9744, pRK5-PRO9821, pRK5-PRO9852,pRK5-PRO9873, pRK5-PRO10196, pRK5-PRO34778, pRK5-PRO20233,pRK5-PRO21956, pRK5-PRO57290, pRK5-PRO38465, pRK5-PRO38683 orpRK5-PRO85161 DNA is added. The cells are first concentrated from thespinner flask by centrifugation and washed with PBS. The DNA-dextranprecipitate is incubated on the cell pellet for four hours. The cellsare treated with 20% glycerol for 90 seconds, washed with tissue culturemedium, and re-introduced into the spinner flask containing tissueculture medium, 5 μg/ml bovine insulin and 0.1 μg/ml bovine transferrin.After about four days, the conditioned media is centrifuged and filteredto remove cells and debris. The sample containing expressed PRO226,PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382, PRO444,PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343, PRO1379,PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571, PRO1572,PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381, PRO4407,PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017, PRO7174,PRO9744, PRO9821, PRO9852, PRO9873, PRO10196, PRO34778, PRO20233,PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161 can then beconcentrated and purified by any selected method, such as dialysisand/or column chromatography.

PRO226, PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382,PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343,PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571,PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381,PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017,PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196, PRO34778,PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161 can beexpressed in CHO cells. The pRK5-PRO226, pRK5-PRO257, pRK5-PRO268,pRK5-PRO290, pRK5-PRO36006, pRK5-PRO363, pRK5-PRO365, pRK5-PRO382,pRK5-PRO444, pRK5-PRO705, pRK5-PRO1071, pRK5-PRO1125, pRK5-PRO1134,pRK5-PRO1155, pRK5-PRO1281, pRK5-PRO1343, pRK5-PRO1379, pRK5-PRO1380,pRK5-PRO1387, pRK5-PRO1419, pRK5-PRO1433, pRK5-PRO1474, pRK5-PRO1550,pRK5-PRO1571, pRK5-PRO1572, pRK5-PRO1759, pRK5-PRO1904, pRK5-PRO35193,pRK5-PRO4341, pRK5-PRO4348, pRK5-PRO4369, pRK5-PRO4381, pRK5-PRO4407,pRK5-PRO4425, pRK5-PRO4985, pRK5-PRO4989, pRK5-PRO5737, pRK5-PRO5800,pRK5-PRO5993, pRK5-PRO6017, pRK5-PRO7174, pRK5-PRO9744, pRK5-PRO9821,pRK5-PRO9852, pRK5-PRO9873, pRK5-PRO10196, pRK5-PRO34778, pRK5-PRO20233,pRK5-PRO21956, pRK5-PRO57290, pRK5-PRO38465, pRK5-PRO38683 orpRK5-PRO85161 can be transfected into CHO cells using known reagentssuch as CaPO₄ or DEAE-dextran. As described above, the cell cultures canbe incubated, and the medium replaced with culture medium (alone) ormedium containing a radiolabel such as ³⁵S-methionine. After determiningthe presence of PRO226, PRO257, PRO268, PRO290, PRO36006, PRO363,PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155,PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474,PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348,PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800,PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196,PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161polypeptide, the culture medium may be replaced with serum free medium.Preferably, the cultures are incubated for about 6 days, and then theconditioned medium is harvested. The medium containing the expressedPRO226, PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382,PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343,PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571,PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381,PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017,PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196, PRO34778,PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161 can then beconcentrated and purified by any selected method.

Epitope-tagged PRO226, PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365,PRO382, PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281,PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550,PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369,PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993,PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196,PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161may also be expressed in host CHO cells. The PRO226, PRO257, PRO268,PRO290, PRO36006, PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071,PRO1125, PRO1134, PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387,PRO1419, PRO1433, PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904,PRO35193, PRO4341, PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985,PRO4989, PRO5737, PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821,PRO9852, PRO9873, PRO10196, PRO34778, PRO20233, PRO21956, PRO57290,PRO38465, PRO38683 or PRO85161 may be subcloned out of the pRK5 vector.The subclone insert can undergo PCR to fuse in frame with a selectedepitope tag such as a poly-his tag into a Baculovirus expression vector.The poly-his tagged PRO226, PRO257, PRO268, PRO290, PRO36006, PRO363,PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155,PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474,PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348,PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800,PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196,PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161insert can then be subcloned into a SV40 driven vector containing aselection marker such as DHFR for selection of stable clones. Finally,the CHO cells can be transfected (as described above) with the SV40driven vector. Labeling may be performed, as described above, to verifyexpression. The culture medium containing the expressed poly-His taggedPRO226, PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382,PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343,PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571,PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381,PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017,PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196, PRO34778,PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161 can then beconcentrated and purified by any selected method, such as byNi²⁺-chelate affinity chromatography.

PRO226, PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382,PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343,PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571,PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381,PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017,PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196, PRO34778,PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161 may also beexpressed in CHO and/or COS cells by a transient expression procedure orin CHO cells by another stable expression procedure.

Stable expression in CHO cells is performed using the followingprocedure. The proteins are expressed as an IgG construct(immunoadhesin), in which the coding sequences for the soluble forms(e.g. extracellular domains) of the respective proteins are fused to anIgG1 constant region sequence containing the hinge, CH2 and CH2 domainsand/or is a poly-His tagged form.

Following PCR amplification, the respective DNAs are subcloned in a CHOexpression vector using standard techniques as described in Ausubel etal., Current Protocols of Molecular Biology, Unit 3.16, John Wiley andSons (1997). CHO expression vectors are constructed to have compatiblerestriction sites 5′ and 3′ of the DNA of interest to allow theconvenient shuttling of cDNA's. The vector used expression in CHO cellsis as described in Lucas et al., Nucl. Acids Res. 24:9 (1774-1779(1996), and uses the SV40 early promoter/enhancer to drive expression ofthe cDNA of interest and dihydrofolate reductase (DHFR). DHFR expressionpermits selection for stable maintenance of the plasmid followingtransfection.

Twelve micrograms of the desired plasmid DNA is introduced intoapproximately 10 million CHO cells using commercially availabletransfection reagents Superfect® (Qiagen), Dosper® or Fugene®(Boehringer Mannheim). The cells are grown as described in Lucas et al.,supra. Approximately 3×10⁷ cells are frozen in an ampule for furthergrowth and production as described below.

The ampules containing the plasmid DNA are thawed by placement intowater bath and mixed by vortexing. The contents are pipetted into acentrifuge tube containing 10 mLs of media and centrifuged at 1000 rpmfor 5 minutes. The supernatant is aspirated and the cells areresuspended in 10 mL of selective media (0.2 μm filtered PS20 with 5%0.2 μm diafiltered fetal bovine serum). The cells are then aliquotedinto a 100 mL spinner containing 90 mL of selective media. After 1-2days, the cells are transferred into a 250 mL spinner filled with 150 mLselective growth medium and incubated at 37° C. After another 2-3 days,250 mL, 500 mL and 2000 mL spinners are seeded with 3×10⁵ cells/mL. Thecell media is exchanged with fresh media by centrifugation andresuspension in production medium. Although any suitable CHO media maybe employed, a production medium described in U.S. Pat. No. 5,122,469,issued Jun. 16, 1992 may actually be used. A 3 L production spinner isseeded at 1.2×10⁶ cells/mL. On day 0, the cell number pH ie determined.On day 1, the spinner is sampled and sparging with filtered air iscommenced. On day 2, the spinner is sampled, the temperature shifted to33° C., and 30 mL of 500 g/L glucose and 0.6 mL of 10% antifoam (e.g.,35% polydimethylsiloxane emulsion, Dow Corning 365 Medical GradeEmulsion) taken. Throughout the production, the pH is adjusted asnecessary to keep it at around 7.2. After 10 days, or until theviability dropped below 70%, the cell culture is harvested bycentrifugation and filtering through a 0.22 μm filter. The filtrate waseither stored at 4° C. or immediately loaded onto columns forpurification.

For the poly-His tagged constructs, the proteins are purified using aNi-NTA column (Qiagen). Before purification, imidazole is added to theconditioned media to a concentration of 5 mM. The conditioned media ispumped onto a 6 ml Ni-NTA column equilibrated in 20 mM Hepes, pH 7.4,buffer containing 0.3 M NaCl and 5 mM imidazole at a flow rate of 4-5ml/min. at 4° C. After loading, the column is washed with additionalequilibration buffer and the protein eluted with equilibration buffercontaining 0.25 M imidazole. The highly purified protein is subsequentlydesalted into a storage buffer containing 10 mM Hepes, 0.14 M NaCl and4% mannitol, pH 6.8, with a 25 ml G25 Superfine (Pharmacia) column andstored at −80° C.

Immunoadhesin (Fc-containing) constructs are purified from theconditioned media as follows. The conditioned medium is pumped onto a 5ml Protein A column (Pharmacia) which had been equilibrated in 20 mM Naphosphate buffer, pH 6.8. After loading, the column is washedextensively with equilibration buffer before elution with 100 mM citricacid, pH 3.5. The eluted protein is immediately neutralized bycollecting 1 ml fractions into tubes containing 275 μL of 1 M Trisbuffer, pH 9. The highly purified protein is subsequently desalted intostorage buffer as described above for the poly-His tagged proteins. Thehomogeneity is assessed by SDS polyacrylamide gels and by N-terminalamino acid sequencing by Edman degradation.

Example 52

Expression of PRO226, PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365,PRO382, PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281,PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550,PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369,PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993,PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196,PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161in Yeast

The following method describes recombinant expression of PRO226, PRO257,PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382, PRO444, PRO705,PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343, PRO1379, PRO1380,PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571, PRO1572, PRO1759,PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381, PRO4407, PRO4425,PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017, PRO7174, PRO9744,PRO9821, PRO9852, PRO9873, PRO10196, PRO34778, PRO20233, PRO21956,PRO57290, PRO38465, PRO38683 or PRO85161 in yeast.

First, yeast expression vectors are constructed for intracellularproduction or secretion of PRO226, PRO257, PRO268, PRO290, PRO36006,PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125, PRO1134,PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433,PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341,PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737,PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873,PRO10196, PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 orPRO85161 from the ADH2/GAPDH promoter. DNA encoding PRO226, PRO257,PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382, PRO444, PRO705,PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343, PRO1379, PRO1380,PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571, PRO1572, PRO1759,PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381, PRO4407, PRO4425,PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017, PRO7174, PRO9744,PRO9821, PRO9852, PRO9873, PRO10196, PRO34778, PRO20233, PRO21956,PRO57290, PRO38465, PRO38683 or PRO85161 and the promoter is insertedinto suitable restriction enzyme sites in the selected plasmid to directintracellular expression of PRO226, PRO257, PRO268, PRO290, PRO36006,PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125, PRO1134,PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433,PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341,PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737,PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873,PRO10196, PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 orPRO85161. For secretion, DNA encoding PRO226, PRO257, PRO268, PRO290,PRO36006, PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125,PRO1134, PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419,PRO1433, PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193,PRO4341, PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989,PRO5737, PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852,PRO9873, PRO10196, PRO34778, PRO20233, PRO21956, PRO57290, PRO38465,PRO38683 or PRO85161 can be cloned into the selected plasmid, togetherwith DNA encoding the ADH2/GAPDH promoter, a native PRO226, PRO257,PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382, PRO444, PRO705,PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343, PRO1379, PRO1380,PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571, PRO1572, PRO1759,PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381, PRO4407, PRO4425,PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017, PRO7174, PRO9744,PRO9821, PRO9852, PRO9873, PRO10196, PRO34778, PRO20233, PRO21956,PRO57290, PRO38465, PRO38683 or PRO85161 signal peptide or othermammalian signal peptide, or, for example, a yeast alpha-factor orinvertase secretory signal/leader sequence, and linker sequences (ifneeded) for expression of PRO226, PRO257, PRO268, PRO290, PRO36006,PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125, PRO1134,PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433,PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341,PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737,PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873,PRO10196, PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 orPRO85161.

Yeast cells, such as yeast strain AB110, can then be transformed withthe expression plasmids described above and cultured in selectedfermentation media. The transformed yeast supernatants can be analyzedby precipitation with 10% trichloroacetic acid and separation bySDS-PAGE, followed by staining of the gels with Coomassie Blue stain.

Recombinant PRO226, PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365,PRO382, PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281,PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550,PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369,PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993,PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196,PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161can subsequently be isolated and purified by removing the yeast cellsfrom the fermentation medium by centrifugation and then concentratingthe medium using selected cartridge filters. The concentrate containingPRO226, PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382,PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343,PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571,PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381,PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017,PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196, PRO34778,PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161 may furtherbe purified using selected column chromatography resins.

Example 53

Expression of PRO226, PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365,PRO382, PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281,PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550,PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369,PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993,PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196,PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161in Baculovirus-Infected Insect Cells

The following method describes recombinant expression of PRO226, PRO257,PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382, PRO444, PRO705,PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343, PRO1379, PRO1380,PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571, PRO1572, PRO1759,PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381, PRO4407, PRO4425,PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017, PRO7174, PRO9744,PRO9821, PRO9852, PRO9873, PRO10196, PRO34778, PRO20233, PRO21956,PRO57290, PRO38465, PRO38683 or PRO85161 in Baculovirus-infected insectcells.

The sequence coding for PRO226, PRO257, PRO268, PRO290, PRO36006,PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125, PRO1134,PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433,PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341,PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737,PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873,PRO10196, PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 orPRO85161 is fused upstream of an epitope tag contained within abaculovirus expression vector. Such epitope tags include poly-his tagsand immunoglobulin tags (like Fc regions of IgG). A variety of plasmidsmay be employed, including plasmids derived from commercially availableplasmids such as pVL1393 (Novagen). Briefly, the sequence encodingPRO226, PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382,PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343,PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571,PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381,PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017,PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196, PRO34778,PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161 or thedesired portion of the coding sequence of PRO226, PRO257, PRO268,PRO290, PRO36006, PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071,PRO1125, PRO1134, PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387,PRO1419, PRO1433, PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904,PRO35193, PRO4341, PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985,PRO4989, PRO5737, PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821,PRO9852, PRO9873, PRO10196, PRO34778, PRO20233, PRO21956, PRO57290,PRO38465, PRO38683 or PRO85161 such as the sequence encoding theextracellular domain of a transmembrane protein or the sequence encodingthe mature protein if the protein is extracellular is amplified by PCRwith primers complementary to the 5′ and 3′ regions. The 5′ primer mayincorporate flanking (selected) restriction enzyme sites. The product isthen digested with those selected restriction enzymes and subcloned intothe expression vector.

Recombinant baculovirus is generated by co-transfecting the aboveplasmid and BaculoGold™ virus DNA (Pharmingen) into Spodopterafrugiperda (“Sf9”) cells (ATCC CRL 1711) using lipofectin (commerciallyavailable from GIBCO-BRL). After 4-5 days of incubation at 28° C., thereleased viruses are harvested and used for further amplifications.Viral infection and protein expression are performed as described byO'Reilley et al., Baculovirus expression vectors: A Laboratory Manual,Oxford: Oxford University Press (1994).

Expressed poly-his tagged PRO226, PRO257, PRO268, PRO290, PRO36006,PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125, PRO1134,PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433,PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341,PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737,PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873,PRO10196, PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 orPRO85161 can then be purified, for example, by Ni²⁺-chelate affinitychromatography as follows. Extracts are prepared from recombinantvirus-infected Sf9 cells as described by Rupert et al., Nature,362:175-179 (1993). Briefly, Sf9 cells are washed, resuspended insonication buffer (25 mL Hepes, pH 7.9; 12.5 mM MgCl₂; 0.1 mM EDTA; 10%glycerol; 0.1% NP-40; 0.4 M KCl), and sonicated twice for 20 seconds onice. The sonicates are cleared by centrifugation, and the supernatant isdiluted 50-fold in loading buffer (50 mM phosphate, 300 mM NaCl, 10%glycerol, pH 7.8) and filtered through a 0.45 μm filter. A Ni²⁺-NTAagarose column (commercially available from Qiagen) is prepared with abed volume of 5 mL, washed with 25 mL of water and equilibrated with 25mL of loading buffer. The filtered cell extract is loaded onto thecolumn at 0.5 mL per minute. The column is washed to baseline A₂₈₀ withloading buffer, at which point fraction collection is started. Next, thecolumn is washed with a secondary wash buffer (50 mM phosphate; 300 mMNaCl, 10% glycerol, pH 6.0), which elutes nonspecifically bound protein.After reaching A₂₈₀ baseline again, the column is developed with a 0 to500 mM Imidazole gradient in the secondary wash buffer. One mL fractionsare collected and analyzed by SDS-PAGE and silver staining or Westernblot with Ni²⁺-NTA-conjugated to alkaline phosphatase (Qiagen).Fractions containing the eluted His₁₀-tagged PRO226, PRO257, PRO268,PRO290, PRO36006, PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071,PRO1125, PRO1134, PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387,PRO1419, PRO1433, PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904,PRO35193, PRO4341, PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985,PRO4989, PRO5737, PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821,PRO9852, PRO9873, PRO10196, PRO34778, PRO20233, PRO21956, PRO57290,PRO38465, PRO38683 or PRO85161 are pooled and dialyzed against loadingbuffer.

Alternatively, purification of the IgG tagged (or Fc tagged) PRO226,PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382, PRO444,PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343, PRO1379,PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571, PRO1572,PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381, PRO4407,PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017, PRO7174,PRO9744, PRO9821, PRO9852, PRO9873, PRO10196, PRO34778, PRO20233,PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161 can be performedusing known chromatography techniques, including for instance, Protein Aor protein G column chromatography.

Example 54 Tissue Expression Profiling Using GeneExpress®

A proprietary database containing gene expression information(GeneExpress®, Gene Logic Inc., Gaithersburg, Md.) was analyzed in anattempt to identify polypeptides (and their encoding nucleic acids)whose expression is significantly upregulated in a particular tumortissue(s) of interest as compared to other tumor(s) and/or normaltissues. Specifically, analysis of the GeneExpress® database wasconducted using either software available through Gene Logic Inc.,Gaithersburg, Md., for use with the GeneExpress® database or withproprietary software written and developed at Genentech, Inc. for usewith the GeneExpress® database. The rating of positive hits in theanalysis is based upon several criteria including, for example, tissuespecificity, tumor specificity and expression level in normal essentialand/or normal proliferating tissues. The following is a list ofmolecules whose tissue expression profile as determined from an analysisof the GeneExpress® database evidences high tissue expression andsignificant upregulation of expression in a specific tumor or tumors ascompared to other tumor(s) and/or normal tissues and optionallyrelatively low expression in normal essential and/or normalproliferating tissues. Tissue expression profiling was performed onseveral UNQ genes the results of which are disclosed in Example 48.

Example 55 Microarray Analysis to Detect Upregulation of UNQ Genes inCancerous Tumors

Nucleic acid microarrays, often containing thousands of gene sequences,are useful for identifying differentially expressed genes in diseasedtissues as compared to their normal counterparts. Using nucleic acidmicroarrays, test and control mRNA samples from test and control tissuesamples are reverse transcribed and labeled to generate cDNA probes. ThecDNA probes are then hybridized to an array of nucleic acids immobilizedon a solid support. The array is configured such that the sequence andposition of each member of the array is known. For example, a selectionof genes known to be expressed in certain disease states may be arrayedon a solid support. Hybridization of a labeled probe with a particulararray member indicates that the sample from which the probe was derivedexpresses that gene. If the hybridization signal of a probe from a test(disease tissue) sample is greater than hybridization signal of a probefrom a control (normal tissue) sample, the gene or genes overexpressedin the disease tissue are identified. The implication of this result isthat an overexpressed protein in a diseased tissue is useful not only asa diagnostic marker for the presence of the disease condition, but alsoas a therapeutic target for treatment of the disease condition.

The methodology of hybridization of nucleic acids and microarraytechnology is well known in the art. In one example, the specificpreparation of nucleic acids for hybridization and probes, slides, andhybridization conditions are all detailed in PCT Patent ApplicationSerial No. PCT/US01/10482, filed on Mar. 30, 2001 and which is hereinincorporated by reference.

In the present example, cancerous tumors derived from various humantissues were studied for upregulated gene expression relative tocancerous tumors from different tissue types and/or non-cancerous humantissues in an attempt to identify those polypeptides which areoverexpressed in a particular cancerous tumor(s). In certainexperiments, cancerous human tumor tissue and non-cancerous human tumortissue of the same tissue type (often from the same patient) wereobtained and analyzed for UNQ polypeptide expression. Additionally,cancerous human tumor tissue from any of a variety of different humantumors was obtained and compared to a “universal” epithelial controlsample which was prepared by pooling non-cancerous human tissues ofepithelial origin, including liver, kidney, and lung. mRNA isolated fromthe pooled tissues represents a mixture of expressed gene products fromthese different tissues. Microarray hybridization experiments using thepooled control samples generated a linear plot in a 2-color analysis.The slope of the line generated in a 2-color analysis was then used tonormalize the ratios of (test:control detection) within each experiment.The normalized ratios from various experiments were then compared andused to identify clustering of gene expression. Thus, the pooled“universal control” sample not only allowed effective relative geneexpression determinations in a simple 2-sample comparison, it alsoallowed multi-sample comparisons across several experiments.

In the present experiments, nucleic acid probes derived from the hereindescribed UNQ polypeptide-encoding nucleic acid sequences were used inthe creation of the microarray and RNA from various tumor tissues wereused for the hybridization thereto. Below is shown the results of theseexperiments, demonstrating that various UNQ polypeptides of the presentinvention are significantly overexpressed in various human tumor tissuesas compared to their normal counterpart tissue(s). Moreover, all of themolecules shown below are significantly overexpressed in their specifictumor tissue(s) as compared to in the “universal” epithelial control. Asdescribed above, these data demonstrate that the UNQ polypeptides of thepresent invention are useful not only as diagnostic markers for thepresence of one or more cancerous tumors, but also serve as therapeutictargets for the treatment of those tumors. Microarray analysis wasperformed on several UNQ genes the results of which are disclosed inExample 48.

Example 56 Quantitative Analysis of UNQ mRNA Expression

In this assay, a 5′ nuclease assay (for example, TaqMan®) and real-timequantitative PCR (for example, ABI Prizm 7700 Sequence Detection System®(Perkin Elmer, Applied Biosystems Division, Foster City, Calif.)), wereused to find genes that are significantly overexpressed in a canceroustumor or tumors as compared to other cancerous tumors or normalnon-cancerous tissue. The 5′ nuclease assay reaction is a fluorescentPCR-based technique which makes use of the 5′ exonuclease activity ofTaq DNA polymerase enzyme to monitor gene expression in real time. Twooligonucleotide primers (whose sequences are based upon the gene or ESTsequence of interest) are used to generate an amplicon typical of a PCRreaction. A third oligonucleotide, or probe, is designed to detectnucleotide sequence located between the two PCR primers. The probe isnon-extendible by Taq DNA polymerase enzyme, and is labeled with areporter fluorescent dye and a quencher fluorescent dye. Anylaser-induced emission from the reporter dye is quenched by thequenching dye when the two dyes are located close together as they areon the probe. During the PCR amplification reaction, the Taq DNApolymerase enzyme cleaves the probe in a template-dependent manner. Theresultant probe fragments disassociate in solution, and signal from thereleased reporter dye is free from the quenching effect of the secondfluorophore. One molecule of reporter dye is liberated for each newmolecule synthesized, and detection of the unquenched reporter dyeprovides the basis for quantitative interpretation of the data.

The 5′ nuclease procedure is run on a real-time quantitative PCR devicesuch as the ABI Prism 7700™ Sequence Detection. The system consists of athermocycler, laser, charge-coupled device (CCD) camera and computer.The system amplifies samples in a 96-well format on a thermocycler.During amplification, laser-induced fluorescent signal is collected inreal-time through fiber optics cables for all 96 wells, and detected atthe CCD. The system includes software for running the instrument and foranalyzing the data.

The starting material for the screen was mRNA isolated from a variety ofdifferent cancerous tissues. The mRNA is quantitated precisely, e.g.,fluorometrically. As a negative control, RNA was isolated from variousnormal tissues of the same tissue type as the cancerous tissues beingtested.

5′ nuclease assay data are initially expressed as Ct, or the thresholdcycle. This is defined as the cycle at which the reporter signalaccumulates above the background level of fluorescence. The ΔCt valuesare used as quantitative measurement of the relative number of startingcopies of a particular target sequence in a nucleic acid sample whencomparing cancer mRNA results to normal human mRNA results. As one Ctunit corresponds to 1 PCR cycle or approximately a 2-fold relativeincrease relative to normal, two units corresponds to a 4-fold relativeincrease, 3 units corresponds to an 8-fold relative increase and so on,one can quantitatively measure the relative fold increase in mRNAexpression between two or more different tissues. Using this technique,the molecules have been identified as being significantly overexpressedin a particular tumor(s) as compared to their normal non-cancerouscounterpart tissue(s) (from both the same and different tissue donors)and thus, represent excellent polypeptide targets for the diagnosis andtherapy of cancer in mammals. Specific results for a UNQ gene aredisclosed in Example 48.

Example 57 In Situ Hybridization

In situ hybridization is a powerful and versatile technique for thedetection and localization of nucleic acid sequences within cell ortissue preparations. It may be useful, for example, to identify sites ofgene expression, analyze the tissue distribution of transcription,identify and localize viral infection, follow changes in specific mRNAsynthesis and aid in chromosome mapping.

In situ hybridization was performed following an optimized version ofthe protocol by Lu and Gillett, Cell Vision 1:169-176 (1994), usingPCR-generated ³³P-labeled riboprobes. Briefly, formalin-fixed,paraffin-embedded human tissues were sectioned, deparaffinized,deproteinated in proteinase K (20 g/ml) for 15 minutes at 37° C., andfurther processed for in situ hybridization as described by Lu andGillett, supra. A [3-P] UTP-labeled antisense riboprobe was generatedfrom a PCR product and hybridized at 55° C. overnight. The slides weredipped in Kodak NTB2 nuclear track emulsion and exposed for 4 weeks.

³³P-Riboprobe Synthesis

6.0 μl (125 mCi) of ³³P-UTP (Amersham BF 1002, SA<2000 Ci/mmol) werespeed vac dried. To each tube containing dried ³³P-UTP, the followingingredients were added:

2.0 μl 5× transcription buffer

1.0 μl DTT (100 mM)

2.0 μl NTP mix (2.5 mM: 101; each of 10 mM GTP, CTP & ATP+10 μl H₂O)

1.0 μl UTP (50 μM)

1.0 μl Rnasin

1.0 μl DNA template (1 μg)

1.0 μl H₂O

1.0 μl RNA polymerase (for PCR products T3=AS, T7=S, usually)

The tubes were incubated at 37° C. for one hour. 1.0 μl RQ1 DNase wereadded, followed by incubation at 37° C. for 15 minutes. 90 μl TE (10 mMTris pH 7.6/1 mM EDTA pH 8.0) were added, and the mixture was pipettedonto DE81 paper. The remaining solution was loaded in a Microcon-50ultrafiltration unit, and spun using program 10 (6 minutes). Thefiltration unit was inverted over a second tube and spun using program 2(3 minutes). After the final recovery spin, 100 μl TE were added. 1 μlof the final product was pipetted on DE81 paper and counted in 6 ml ofBiofluor II.

The probe was run on a TBE/urea gel. 1-3 μl of the probe or 5 μl of RNAMrk III were added to 3 μl of loading buffer. After heating on a 95° C.heat block for three minutes, the probe was immediately placed on ice.The wells of gel were flushed, the sample loaded, and run at 180-250volts for 45 minutes. The gel was wrapped in saran wrap and exposed toXAR film with an intensifying screen in −70° C. freezer one hour toovernight.

³³P-Hybridization

A. Pretreatment of Frozen Sections

The slides were removed from the freezer, placed on aluminium trays andthawed at room temperature for 5 minutes. The trays were placed in 55°C. incubator for five minutes to reduce condensation. The slides werefixed for 10 minutes in 4% paraformaldehyde on ice in the fume hood, andwashed in 0.5×SSC for 5 minutes, at room temperature (25 ml 20×SSC+975ml SQ H₂O). After deproteination in 0.5 μg/ml proteinase K for 10minutes at 37° C. (12.5 μl of 10 mg/ml stock in 250 ml prewarmedRNase-free RNAse buffer), the sections were washed in 0.5×SSC for 10minutes at room temperature. The sections were dehydrated in 70%, 95%,100% ethanol, 2 minutes each.

B. Pretreatment of Paraffin-Embedded Sections

The slides were deparaffinized, placed in SQ H₂O, and rinsed twice in2×SSC at room temperature, for 5 minutes each time. The sections weredeproteinated in 20 μg/ml proteinase K (500 μl of 10 mg/ml in 250 mlRNase-free RNase buffer; 37° C., 15 minutes)—human embryo, or 8×proteinase K (100 μl in 250 ml Rnase buffer, 37° C., 30minutes)—formalin tissues. Subsequent rinsing in 0.5×SSC and dehydrationwere performed as described above.

C. Prehybridization

The slides were laid out in a plastic box lined with Box buffer (4×SSC,50% formamide)—saturated filter paper.

D. Hybridization

1.0×10⁶ cpm probe and 1.0 μl tRNA (50 mg/ml stock) per slide were heatedat 95° C. for 3 minutes. The slides were cooled on ice, and 48 μlhybridization buffer were added per slide. After vortexing, 50 μl ³³Pmix were added to 50 μl prehybridization on slide. The slides wereincubated overnight at 55° C.

E. Washes

Washing was done 2×10 minutes with 2×SSC, EDTA at room temperature (400ml 20×SSC+16 ml 0.25M EDTA, V_(f)=4 L), followed by RNaseA treatment at37° C. for 30 minutes (500 μl of 10 mg/ml in 250 ml Rnase buffer=20μg/ml), The slides were washed 2×10 minutes with 2×SSC, EDTA at roomtemperature. The stringency wash conditions were as follows: 2 hours at55° C., 0.1×SSC, EDTA (20 ml 20×SSC+16 ml EDTA, V_(f)=4 L).

F. Oligonucleotides

In situ analysis was performed on a variety of DNA sequences disclosedherein. The oligonucleotides employed for these analyses were obtainedso as to be complementary to the nucleic acids (or the complementsthereof) as shown in the accompanying figures.

G. Results

In situ analysis was performed on a variety of DNA sequences disclosedherein the results of which are disclosed in Example 48.

Example 58

Preparation of Antibodies that Bind PRO226, PRO257, PRO268, PRO290,PRO36006, PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125,PRO1134, PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419,PRO1433, PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193,PRO4341, PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989,PRO5737, PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852,PRO9873, PRO10196, PRO34778, PRO20233, PRO21956, PRO57290, PRO38465,PRO38683 or PRO85161

This example illustrates preparation of monoclonal antibodies which canspecifically bind PRO226, PRO257, PRO268, PRO290, PRO36006, PRO363,PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155,PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474,PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348,PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800,PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196,PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161.

Techniques for producing the monoclonal antibodies are known in the artand are described, for instance, in Goding, supra. Immunogens that maybe employed include purified PRO226, PRO257, PRO268, PRO290, PRO36006,PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125, PRO1134,PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433,PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341,PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737,PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873,PRO10196, PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 orPRO85161 polypeptides, fusion proteins containing PRO226, PRO257,PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382, PRO444, PRO705,PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343, PRO1379, PRO1380,PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571, PRO1572, PRO1759,PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381, PRO4407, PRO4425,PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017, PRO7174, PRO9744,PRO9821, PRO9852, PRO9873, PRO10196, PRO34778, PRO20233, PRO21956,PRO57290, PRO38465, PRO38683 or PRO85161 polypeptides, and cellsexpressing recombinant PRO226, PRO257, PRO268, PRO290, PRO36006, PRO363,PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155,PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474,PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348,PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800,PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196,PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161polypeptides on the cell surface. Selection of the immunogen can be madeby the skilled artisan without undue experimentation.

Mice, such as Balb/c, are immunized with the PRO226, PRO257, PRO268,PRO290, PRO36006, PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071,PRO1125, PRO1134, PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387,PRO1419, PRO1433, PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904,PRO35193, PRO4341, PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985,PRO4989, PRO5737, PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821,PRO9852, PRO9873, PRO10196, PRO34778, PRO20233, PRO21956, PRO57290,PRO38465, PRO38683 or PRO85161 immunogen emulsified in complete Freund'sadjuvant and injected subcutaneously or intraperitoneally in an amountfrom 1-100 micrograms. Alternatively, the immunogen is emulsified inMPL-TDM adjuvant (Ribi Immunochemical Research, Hamilton, Mont.) andinjected into the animal's hind foot pads. The immunized mice are thenboosted 10 to 12 days later with additional immunogen emulsified in theselected adjuvant. Thereafter, for several weeks, the mice may also beboosted with additional immunization injections. Serum samples may beperiodically obtained from the mice by retro-orbital bleeding fortesting in ELISA assays to detect anti-PRO226, anti-PRO257, anti-PRO268,anti-PRO290, anti-PRO36006, anti-PRO363, anti-PRO365, anti-PRO382,anti-PRO444, anti-PRO705, anti-PRO1071, anti-PRO1125, anti-PRO1134,anti-PRO1155, anti-PRO1281, anti-PRO1343, anti-PRO1379, anti-PRO1380,anti-PRO1387, anti-PRO1419, anti-PRO1433, anti-PRO1474, anti-PRO1550,anti-PRO1571, anti-PRO1572, anti-PRO1759, anti-PRO1904, anti-PRO35193,anti-PRO4341, anti-PRO4348, anti-PRO4369, anti-PRO4381, anti-PRO4407,anti-PRO4425, anti-PRO4985, anti-PRO4989, anti-PRO5737, anti-PRO5800,anti-PRO5993, anti-PRO6017, anti-PRO7174, anti-PRO9744, anti-PRO9821,anti-PRO9852, anti-PRO9873, anti-PRO10196, anti-PRO34778, anti-PRO20233,anti-PRO21956, anti-PRO57290, anti-PRO38465, anti-PRO38683 oranti-PRO85161 antibodies.

After a suitable antibody titer has been detected, the animals“positive” for antibodies can be injected with a final intravenousinjection of PRO226, PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365,PRO382, PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281,PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550,PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369,PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993,PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196,PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161.Three to four days later, the mice are sacrificed and the spleen cellsare harvested. The spleen cells are then fused (using 35% polyethyleneglycol) to a selected murine myeloma cell line such as P3X63AgU.1,available from ATCC, No. CRL 1597. The fusions generate hybridoma cellswhich can then be plated in 96 well tissue culture plates containing HAT(hypoxanthine, aminopterin, and thymidine) medium to inhibitproliferation of non-fused cells, myeloma hybrids, and spleen cellhybrids.

The hybridoma cells will be screened in an ELISA for reactivity againstPRO226, PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382,PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343,PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571,PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381,PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017,PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196, PRO34778,PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161.Determination of “positive” hybridoma cells secreting the desiredmonoclonal antibodies against PRO226, PRO257, PRO268, PRO290, PRO36006,PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125, PRO1134,PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433,PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341,PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737,PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873,PRO10196, PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 orPRO85161 is within the skill in the art.

The positive hybridoma cells can be injected intraperitoneally intosyngeneic Balb/c mice to produce ascites containing the anti-PRO226,anti-PRO257, anti-PRO268, anti-PRO290, anti-PRO36006, anti-PRO363,anti-PRO365, anti-PRO382, anti-PRO444, anti-PRO705, anti-PRO1071,anti-PRO1125, anti-PRO1134, anti-PRO1155, anti-PRO1281, anti-PRO1343,anti-PRO1379, anti-PRO1380, anti-PRO1387, anti-PRO1419, anti-PRO1433,anti-PRO1474, anti-PRO1550, anti-PRO1571, anti-PRO1572, anti-PRO1759,anti-PRO1904, anti-PRO35193, anti-PRO4341, anti-PRO4348, anti-PRO4369,anti-PRO4381, anti-PRO4407, anti-PRO4425, anti-PRO4985, anti-PRO4989,anti-PRO5737, anti-PRO5800, anti-PRO5993, anti-PRO6017, anti-PRO7174,anti-PRO9744, anti-PRO9821, anti-PRO9852, anti-PRO9873, anti-PRO10196,anti-PRO34778, anti-PRO20233, anti-PRO21956, anti-PRO57290,anti-PRO38465, anti-PRO38683 or anti-PRO85161 monoclonal antibodies.Alternatively, the hybridoma cells can be grown in tissue culture flasksor roller bottles. Purification of the monoclonal antibodies produced inthe ascites can be accomplished using ammonium sulfate precipitation,followed by gel exclusion chromatography. Alternatively, affinitychromatography based upon binding of antibody to protein A or protein Gcan be employed.

Example 59

Purification of PRO226, PRO257, PRO268, PRO290, PRO36006, PRO363,PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155,PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474,PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348,PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800,PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196,PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161Polypeptides Using Specific Antibodies

Native or recombinant PRO226, PRO257, PRO268, PRO290, PRO36006, PRO363,PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155,PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474,PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348,PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800,PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196,PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161polypeptides may be purified by a variety of standard techniques in theart of protein purification. For example, pro-PRO226, pro-PRO257,pro-PRO268, pro-PRO290, pro-PRO36006, pro-PRO363, pro-PRO365,pro-PRO382, pro-PRO444, pro-PRO705, pro-PRO1071, pro-PRO1125,pro-PRO1134, pro-PRO1155, pro-PRO1281, pro-PRO1343, pro-PRO1379,pro-PRO1380, pro-PRO1387, pro-PRO1419, pro-PRO1433, pro-PRO1474,pro-PRO1550, pro-PRO1571, pro-PRO1572, pro-PRO1759, pro-PRO1904,pro-PRO35193, pro-PRO4341, pro-PRO4348, pro-PRO4369, pro-PRO4381,pro-PRO4407, pro-PRO4425, pro-PRO4985, pro-PRO4989, pro-PRO5737,pro-PRO5800, pro-PRO5993, pro-PRO6017, pro-PRO7174, pro-PRO9744,pro-PRO9821, pro-PRO9852, pro-PRO9873, pro-PRO10196, pro-PRO34778,pro-PRO20233, pro-PRO21956, pro-PRO57290, pro-PRO38465, pro-PRO38683 orpro-PRO85161 polypeptide, mature PRO226, mature PRO257, mature PRO268,mature PRO290, mature PRO36006, mature PRO363, mature PRO365, maturePRO382, mature PRO444, mature PRO705, mature PRO1071, mature PRO1125,mature PRO1134, mature PRO1155, mature PRO1281, mature PRO1343, maturePRO1379, mature PRO1380, mature PRO1387, mature PRO1419, mature PRO1433,mature PRO1474, mature PRO1550, mature PRO1571, mature PRO1572, maturePRO1759, mature PRO1904, mature PRO35193, mature PRO4341, maturePRO4348, mature PRO4369, mature PRO4381, mature PRO4407, mature PRO4425,mature PRO4985, mature PRO4989, mature PRO5737, mature PRO5800, maturePRO5993, mature PRO6017, mature PRO7174, mature PRO9744, mature PRO9821,mature PRO9852, mature PRO9873, mature PRO10196, mature PRO34778, maturePRO20233, mature PRO21956, mature PRO57290, mature PRO38465, maturePRO38683 or mature PRO85161 polypeptide, or pre-PRO226, pre-PRO257,pre-PRO268, pre-PRO290, pre-PRO36006, pre-PRO363, pre-PRO365,pre-PRO382, pre-PRO444, pre-PRO705, pre-PRO1071, pre-PRO1125,pre-PRO1134, pre-PRO1155, pre-PRO1281, pre-PRO1343, pre-PRO1379,pre-PRO1380, pre-PRO1387, pre-PRO1419, pre-PRO1433, pre-PRO1474,pre-PRO1550, pre-PRO1571, pre-PRO1572, pre-PRO1759, pre-PRO1904,pre-PRO35193, pre-PRO4341, pre-PRO4348, pre-PRO4369, pre-PRO4381,pre-PRO4407, pre-PRO4425, pre-PRO4985, pre-PRO4989, pre-PRO5737,pre-PRO5800, pre-PRO5993, pre-PRO6017, pre-PRO7174, pre-PRO9744,pre-PRO9821, pre-PRO9852, pre-PRO9873, pre-PRO10196, pre-PRO34778,pre-PRO20233, pre-PRO21956, pre-PRO57290, pre-PRO38465, pre-PRO38683 orpre-PRO85161 polypeptide is purified by immunoaffinity chromatographyusing antibodies specific for the PRO226, PRO257, PRO268, PRO290,PRO36006, PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125,PRO1134, PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419,PRO1433, PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193,PRO4341, PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989,PRO5737, PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852,PRO9873, PRO10196, PRO34778, PRO20233, PRO21956, PRO57290, PRO38465,PRO38683 or PRO85161 polypeptide of interest. In general, animmunoaffinity column is constructed by covalently coupling theanti-PRO226, anti-PRO257, anti-PRO268, anti-PRO290, anti-PRO36006,anti-PRO363, anti-PRO365, anti-PRO382, anti-PRO444, anti-PRO705,anti-PRO1071, anti-PRO1125, anti-PRO1134, anti-PRO1155, anti-PRO1281,anti-PRO1343, anti-PRO1379, anti-PRO1380, anti-PRO1387, anti-PRO1419,anti-PRO1433, anti-PRO1474, anti-PRO1550, anti-PRO1571, anti-PRO1572,anti-PRO1759, anti-PRO1904, anti-PRO35193, anti-PRO4341, anti-PRO4348,anti-PRO4369, anti-PRO4381, anti-PRO4407, anti-PRO4425, anti-PRO4985,anti-PRO4989, anti-PRO5737, anti-PRO5800, anti-PRO5993, anti-PRO6017,anti-PRO7174, anti-PRO9744, anti-PRO9821, anti-PRO9852, anti-PRO9873,anti-PRO10196, anti-PRO34778, anti-PRO20233, anti-PRO21956,anti-PRO57290, anti-PRO38465, anti-PRO38683 or anti-PRO85161 polypeptideantibody to an activated chromatographic resin.

Polyclonal immunoglobulins are prepared from immune sera either byprecipitation with ammonium sulfate or by purification on immobilizedProtein A (Pharmacia LKB Biotechnology, Piscataway, N.J.). Likewise,monoclonal antibodies are prepared from mouse ascites fluid by ammoniumsulfate precipitation or chromatography on immobilized Protein A.Partially purified immunoglobulin is covalently attached to achromatographic resin such as CnBr-activated SEPHAROSE™ (Pharmacia LKBBiotechnology). The antibody is coupled to the resin, the resin isblocked, and the derivative resin is washed according to themanufacturer's instructions.

Such an immunoaffinity column is utilized in the purification of PRO226,PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382, PRO444,PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343, PRO1379,PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571, PRO1572,PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381, PRO4407,PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017, PRO7174,PRO9744, PRO9821, PRO9852, PRO9873, PRO10196, PRO34778, PRO20233,PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161 polypeptide bypreparing a fraction from cells containing PRO226, PRO257, PRO268,PRO290, PRO36006, PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071,PRO1125, PRO1134, PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387,PRO1419, PRO1433, PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904,PRO35193, PRO4341, PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985,PRO4989, PRO5737, PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821,PRO9852, PRO9873, PRO10196, PRO34778, PRO20233, PRO21956, PRO57290,PRO38465, PRO38683 or PRO85161 polypeptide in a soluble form. Thispreparation is derived by solubilization of the whole cell or of asubcellular fraction obtained via differential centrifugation by theaddition of detergent or by other methods well known in the art.Alternatively, soluble polypeptide containing a signal sequence may besecreted in useful quantity into the medium in which the cells aregrown.

A soluble PRO226, PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365,PRO382, PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281,PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550,PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369,PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993,PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196,PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161polypeptide-containing preparation is passed over the immunoaffinitycolumn, and the column is washed under conditions that allow thepreferential absorbance of PRO226, PRO257, PRO268, PRO290, PRO36006,PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125, PRO1134,PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433,PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341,PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737,PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873,PRO10196, PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 orPRO85161 polypeptide (e.g., high ionic strength buffers in the presenceof detergent). Then, the column is eluted under conditions that disruptantibody/PRO226, antibody/PRO257, antibody/PRO268, antibody/PRO290,antibody/PRO36006, antibody/PRO363, antibody/PRO365, antibody/PRO382,antibody/PRO444, antibody/PRO705, antibody/PRO1071, antibody/PRO1125,antibody/PRO1134, antibody/PRO1155, antibody/PRO1281, antibody/PRO1343,antibody/PRO1379, antibody/PRO1380, antibody/PRO1387, antibody/PRO1419,antibody/PRO1433, antibody/PRO1474, antibody/PRO1550, antibody/PRO1571,antibody/PRO1572, antibody/PRO1759, antibody/PRO1904, antibody/PRO35193,antibody/PRO4341, antibody/PRO4348, antibody/PRO4369, antibody/PRO4381,antibody/PRO4407, antibody/PRO4425, antibody/PRO4985, antibody/PRO4989,antibody/PRO5737, antibody/PRO5800, antibody/PRO5993, antibody/PRO6017,antibody/PRO7174, antibody/PRO9744, antibody/PRO9821, antibody/PRO9852,antibody/PRO9873, antibody/PRO10196, antibody/PRO34778,antibody/PRO20233, antibody/PRO21956, antibody/PRO57290,antibody/PRO38465, antibody/PRO38683 or antibody/PRO85161 polypeptidebinding (e.g., a low pH buffer such as approximately pH 2-3, or a highconcentration of a chaotrope such as urea or thiocyanate ion), andPRO226, PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382,PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343,PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571,PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381,PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017,PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196, PRO34778,PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161 polypeptideis collected.

Example 60 Drug Screening

This invention is particularly useful for screening compounds by usingPRO226, PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382,PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343,PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571,PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381,PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017,PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196, PRO34778,PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161polypeptides or binding fragment thereof in any of a variety of drugscreening techniques. The PRO226, PRO257, PRO268, PRO290, PRO36006,PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125, PRO1134,PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433,PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341,PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737,PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873,PRO10196, PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 orPRO85161 polypeptide or fragment employed in such a test may either befree in solution, affixed to a solid support, borne on a cell surface,or located intracellularly. One method of drug screening utilizeseukaryotic or prokaryotic host cells which are stably transformed withrecombinant nucleic acids expressing the PRO226, PRO257, PRO268, PRO290,PRO36006, PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125,PRO1134, PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419,PRO1433, PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193,PRO4341, PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989,PRO5737, PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852,PRO9873, PRO10196, PRO34778, PRO20233, PRO21956, PRO57290, PRO38465,PRO38683 or PRO85161 polypeptide or fragment. Drugs are screened againstsuch transformed cells in competitive binding assays. Such cells, eitherinviable or fixed form, can be used for standard binding assays. One maymeasure, for example, the formation of complexes between PRO226, PRO257,PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382, PRO444, PRO705,PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343, PRO1379, PRO1380,PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571, PRO1572, PRO1759,PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381, PRO4407, PRO4425,PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017, PRO7174, PRO9744,PRO9821, PRO9852, PRO9873, PRO10196, PRO34778, PRO20233, PRO21956,PRO57290, PRO38465, PRO38683 or PRO85161 polypeptide or a fragment andthe agent being tested. Alternatively, one can examine the diminution incomplex formation between the PRO226, PRO257, PRO268, PRO290, PRO36006,PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125, PRO1134,PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433,PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341,PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737,PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873,PRO10196, PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 orPRO85161 polypeptide and its target cell or target receptors caused bythe agent being tested.

Thus, the present invention provides methods of screening for drugs orany other agents which can affect a PRO226, PRO257, PRO268, PRO290,PRO36006, PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125,PRO1134, PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419,PRO1433, PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193,PRO4341, PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989,PRO5737, PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852,PRO9873, PRO10196, PRO34778, PRO20233, PRO21956, PRO57290, PRO38465,PRO38683 or PRO85161 polypeptide-associated disease or disorder. Thesemethods comprise contacting such an agent with an PRO226, PRO257,PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382, PRO444, PRO705,PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343, PRO1379, PRO1380,PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571, PRO1572, PRO1759,PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381, PRO4407, PRO4425,PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017, PRO7174, PRO9744,PRO9821, PRO9852, PRO9873, PRO10196, PRO34778, PRO20233, PRO21956,PRO57290, PRO38465, PRO38683 or PRO85161 polypeptide or fragment thereofand assaying (I) for the presence of a complex between the agent and thePRO226, PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382,PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343,PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571,PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381,PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017,PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196, PRO34778,PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161 polypeptideor fragment, or (ii) for the presence of a complex between the PRO226,PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382, PRO444,PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343, PRO1379,PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571, PRO1572,PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381, PRO4407,PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017, PRO7174,PRO9744, PRO9821, PRO9852, PRO9873, PRO10196, PRO34778, PRO20233,PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161 polypeptide orfragment and the cell, by methods well known in the art. In suchcompetitive binding assays, the PRO226, PRO257, PRO268, PRO290,PRO36006, PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125,PRO1134, PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419,PRO1433, PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193,PRO4341, PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989,PRO5737, PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852,PRO9873, PRO10196, PRO34778, PRO20233, PRO21956, PRO57290, PRO38465,PRO38683 or PRO85161 polypeptide or fragment is typically labeled. Aftersuitable incubation, free PRO226, PRO257, PRO268, PRO290, PRO36006,PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125, PRO1134,PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433,PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341,PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737,PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873,PRO10196, PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 orPRO85161 polypeptide or fragment is separated from that present in boundform, and the amount of free or uncomplexed label is a measure of theability of the particular agent to bind to PRO226, PRO257, PRO268,PRO290, PRO36006, PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071,PRO1125, PRO1134, PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387,PRO1419, PRO1433, PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904,PRO35193, PRO4341, PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985,PRO4989, PRO5737, PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821,PRO9852, PRO9873, PRO10196, PRO34778, PRO20233, PRO21956, PRO57290,PRO38465, PRO38683 or PRO85161 polypeptide or to interfere with thePRO226, PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382,PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343,PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571,PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381,PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017,PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196, PRO34778,PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161polypeptide/cell complex.

Another technique for drug screening provides high throughput screeningfor compounds having suitable binding affinity to a polypeptide and isdescribed in detail in WO 84/03564, published on Sep. 13, 1984. Brieflystated, large numbers of different small peptide test compounds aresynthesized on a solid substrate, such as plastic pins or some othersurface. As applied to a PRO226, PRO257, PRO268, PRO290, PRO36006,PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125, PRO1134,PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433,PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341,PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737,PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873,PRO10196, PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 orPRO85161 polypeptide, the peptide test compounds are reacted withPRO226, PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382,PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343,PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571,PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381,PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017,PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196, PRO34778,PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161 polypeptideand washed. Bound PRO226, PRO257, PRO268, PRO290, PRO36006, PRO363,PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155,PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474,PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348,PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800,PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196,PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161polypeptide is detected by methods well known in the art. PurifiedPRO226, PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382,PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343,PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571,PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381,PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017,PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196, PRO34778,PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161 polypeptidecan also be coated directly onto plates for use in the aforementioneddrug screening techniques. In addition, non-neutralizing antibodies canbe used to capture the peptide and immobilize it on the solid support.

This invention also contemplates the use of competitive drug screeningassays in which neutralizing antibodies capable of binding PRO226,PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382, PRO444,PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343, PRO1379,PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571, PRO1572,PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381, PRO4407,PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017, PRO7174,PRO9744, PRO9821, PRO9852, PRO9873, PRO10196, PRO34778, PRO20233,PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161 polypeptidespecifically compete with a test compound for binding to PRO226, PRO257,PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382, PRO444, PRO705,PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343, PRO1379, PRO1380,PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571, PRO1572, PRO1759,PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381, PRO4407, PRO4425,PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017, PRO7174, PRO9744,PRO9821, PRO9852, PRO9873, PRO10196, PRO34778, PRO20233, PRO21956,PRO57290, PRO38465, PRO38683 or PRO85161 polypeptide or fragmentsthereof. In this manner, the antibodies can be used to detect thepresence of any peptide which shares one or more antigenic determinantswith PRO226, PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382,PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343,PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571,PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381,PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017,PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196, PRO34778,PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161polypeptide.

Example 61 Rational Drug Design

The goal of rational drug design is to produce structural analogs ofbiologically active polypeptide of interest (i.e., a PRO226, PRO257,PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382, PRO444, PRO705,PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343, PRO1379, PRO1380,PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571, PRO1572, PRO1759,PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381, PRO4407, PRO4425,PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017, PRO7174, PRO9744,PRO9821, PRO9852, PRO9873, PRO10196, PRO34778, PRO20233, PRO21956,PRO57290, PRO38465, PRO38683 or PRO85161 polypeptide) or of smallmolecules with which they interact, e.g., agonists, antagonists, orinhibitors. Any of these examples can be used to fashion drugs which aremore active or stable forms of the PRO226, PRO257, PRO268, PRO290,PRO36006, PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125,PRO1134, PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419,PRO1433, PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193,PRO4341, PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989,PRO5737, PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852,PRO9873, PRO10196, PRO34778, PRO20233, PRO21956, PRO57290, PRO38465,PRO38683 or PRO85161 polypeptide or which enhance or interfere with thefunction of the PRO226, PRO257, PRO268, PRO290, PRO36006, PRO363,PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125, PRO1134, PRO1155,PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433, PRO1474,PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341, PRO4348,PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737, PRO5800,PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873, PRO10196,PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161polypeptide in vivo (c.f, Hodgson, Bio/Technology, 2: 19-21 (1991)).

In one approach, the three-dimensional structure of the PRO226, PRO257,PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382, PRO444, PRO705,PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343, PRO1379, PRO1380,PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571, PRO1572, PRO1759,PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381, PRO4407, PRO4425,PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017, PRO7174, PRO9744,PRO9821, PRO9852, PRO9873, PRO10196, PRO34778, PRO20233, PRO21956,PRO57290, PRO38465, PRO38683 or PRO85161 polypeptide, or of a PRO226,PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382, PRO444,PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343, PRO1379,PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571, PRO1572,PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381, PRO4407,PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017, PRO7174,PRO9744, PRO9821, PRO9852, PRO9873, PRO10196, PRO34778, PRO20233,PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161 polypeptide-inhibitorcomplex, is determined by x-ray crystallography, by computer modelingor, most typically, by a combination of the two approaches. Both theshape and charges of the PRO226, PRO257, PRO268, PRO290, PRO36006,PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071, PRO1125, PRO1134,PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387, PRO1419, PRO1433,PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904, PRO35193, PRO4341,PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985, PRO4989, PRO5737,PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821, PRO9852, PRO9873,PRO10196, PRO34778, PRO20233, PRO21956, PRO57290, PRO38465, PRO38683 orPRO85161 polypeptide must be ascertained to elucidate the structure andto determine active site(s) of the molecule. Less often, usefulinformation regarding the structure of the PRO226, PRO257, PRO268,PRO290, PRO36006, PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071,PRO1125, PRO1134, PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387,PRO1419, PRO1433, PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904,PRO35193, PRO4341, PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985,PRO4989, PRO5737, PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821,PRO9852, PRO9873, PRO10196, PRO34778, PRO20233, PRO21956, PRO57290,PRO38465, PRO38683 or PRO85161 polypeptide may be gained by modelingbased on the structure of homologous proteins. In both cases, relevantstructural information is used to design analogous PRO226, PRO257,PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382, PRO444, PRO705,PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343, PRO1379, PRO1380,PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571, PRO1572, PRO1759,PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381, PRO4407, PRO4425,PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017, PRO7174, PRO9744,PRO9821, PRO9852, PRO9873, PRO10196, PRO34778, PRO20233, PRO21956,PRO57290, PRO38465, PRO38683 or PRO85161 polypeptide-like molecules orto identify efficient inhibitors. Useful examples of rational drugdesign may include molecules which have improved activity or stabilityas shown by Braxton and Wells, Biochemistry, 31:7796-7801 (1992) orwhich act as inhibitors, agonists, or antagonists of native peptides asshown by Athauda et al., J. Biochem., 113:742-746 (1993).

It is also possible to isolate a target-specific antibody, selected byfunctional assay, as described above, and then to solve its crystalstructure. This approach, in principle, yields a pharmacore upon whichsubsequent drug design can be based. It is possible to bypass proteincrystallography altogether by generating anti-idiotypic antibodies(anti-ids) to a functional, pharmacologically active antibody. As amirror image of a mirror image, the binding site of the anti-ids wouldbe expected to be an analog of the original receptor. The anti-id couldthen be used to identify and isolate peptides from banks of chemicallyor biologically produced peptides. The isolated peptides would then actas the pharmacore.

By virtue of the present invention, sufficient amounts of the PRO226,PRO257, PRO268, PRO290, PRO36006, PRO363, PRO365, PRO382, PRO444,PRO705, PRO1071, PRO1125, PRO1134, PRO1155, PRO1281, PRO1343, PRO1379,PRO1380, PRO1387, PRO1419, PRO1433, PRO1474, PRO1550, PRO1571, PRO1572,PRO1759, PRO1904, PRO35193, PRO4341, PRO4348, PRO4369, PRO4381, PRO4407,PRO4425, PRO4985, PRO4989, PRO5737, PRO5800, PRO5993, PRO6017, PRO7174,PRO9744, PRO9821, PRO9852, PRO9873, PRO10196, PRO34778, PRO20233,PRO21956, PRO57290, PRO38465, PRO38683 or PRO85161 polypeptide may bemade available to perform such analytical studies as X-raycrystallography. In addition, knowledge of the PRO226, PRO257, PRO268,PRO290, PRO36006, PRO363, PRO365, PRO382, PRO444, PRO705, PRO1071,PRO1125, PRO1134, PRO1155, PRO1281, PRO1343, PRO1379, PRO1380, PRO1387,PRO1419, PRO1433, PRO1474, PRO1550, PRO1571, PRO1572, PRO1759, PRO1904,PRO35193, PRO4341, PRO4348, PRO4369, PRO4381, PRO4407, PRO4425, PRO4985,PRO4989, PRO5737, PRO5800, PRO5993, PRO6017, PRO7174, PRO9744, PRO9821,PRO9852, PRO9873, PRO10196, PRO34778, PRO20233, PRO21956, PRO57290,PRO38465, PRO38683 or PRO85161 polypeptide amino acid sequence providedherein will provide guidance to those employing computer modelingtechniques in place of or in addition to x-ray crystallography.

1. A method of identifying an agent that modulates a phenotypeassociated with a disruption of a gene which encodes for a polypeptide,the method comprising: (a) providing a non-human transgenic animal whosegenome comprises a disruption of a gene which is an ortholog of a humangene that encodes for the polypeptide (SEQ ID NO:68) and which, comparedwith gender matched wild-type littermates, exhibits a phenotypeassociated with said gene disruption, said phenotype comprising at leastone of the following physiological characteristics: increased percentageof CD4 cells in peripheral blood, increased TCRbeta+ in thymus,increased CD11b+CD11c+ in lymph nodes, increased natural killer cells inlymph nodes, increased percentage of CD 117+ cells in peritoneal lavage,decreased IgG2a mean serum levels and increased IgA mean serum levels;(b) measuring a physiological characteristic of the non-human transgenicanimal of (a); (c) comparing the measured physiological characteristicof (b) with that of a gender matched wild-type animal, wherein thephysiological characteristic of the non-human transgenic animal thatdiffers from the physiological characteristic of the wild-type animal isidentified as a phenotype resulting from the gene disruption in thenon-human transgenic animal; (d) administering a test agent to thenon-human transgenic animal of (a); and (e) determining whether the testagent modulates the identified phenotype associated with gene disruptionin the non-human transgenic animal.
 2. The method of claim 1, whereinthe phenotype associated with the gene disruption comprises animmunological disorder.
 3. The method of claim 2, wherein theimmunological disorders are systemic lupus erythematosis; rheumatoidarthritis; juvenile chronic arthritis; spondyloarthropathies; systemicsclerosis (scleroderma); idiopathic inflammatory myopathies(dermatomyositis, polymyositis); Sjogren's syndrome; systemicvasculitis; sarcoidosis; autoimmune hemolytic anemia (immunepancytopenia, paroxysmal nocturnal hemoglobinuria); autoimmunethrombocytopenia (idiopathic thrombocytopenic purpura, immune-mediatedthrombocytopenia); thyroiditis (Grave's disease, Hashimoto'sthyroiditis, juvenile lymphocytic thyroiditis, atrophic thyroiditis);diabetes mellitus; immune-mediated renal disease (glomerulonephritis,tubulointerstitial nephritis); demyelinating diseases of the central andperipheral nervous systems such as multiple sclerosis, idiopathicdemyelinating polyneuropathy or Guillain-Barre syndrome, and chronicinflammatory demyelinating polyneuropathy; hepatobiliary diseases suchas infectious hepatitis (hepatitis A, B, C, D, E and othernon-hepatotropic viruses), autoimmune chronic active hepatitis, primarybiliary cirrhosis, granulomatous hepatitis, and sclerosing cholangitis;inflammatory bowel disease (ulcerative colitis: Crohn's disease);gluten-sensitive enteropathy, and Whipple's disease; autoimmune orimmune-mediated skin diseases including bullous skin diseases, erythemamultiforme and contact dermatitis, psoriasis; allergic diseases such asasthma, allergic rhinitis, atopic dermatitis, food hypersensitivity andurticaria; immunologic diseases of the lung such as eosinophilicpneumonia, idiopathic pulmonary fibrosis and hypersensitivitypneumonitis; or transplantation-associated diseases including graftrejection and graft-versus-host disease.